Stable aqueous formulations of adalimumab

ABSTRACT

The invention provides aqueous pharmaceutical adalimumab compositions suitable for long-term storage of adalimumab, methods of manufacture of these compositions, methods of administration, and kits containing same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/730,399, filed on Oct. 11, 2017, which is acontinuation of U.S. patent application Ser. No. 14/020,733, filed onSep. 6, 2013, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/698,138, filed Sep. 7, 2012, U.S. ProvisionalPatent Application Ser. No. 61/769,581, filed Feb. 26, 2013, and U.S.Provisional Patent Application Ser. No. 61/770,421, filed Feb. 28, 2013,each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to aqueous pharmaceutical compositionssuitable for long-term storage of adalimumab (including antibodyproteins considered or intended as “biosimilar” or “bio-better” variantsof commercially available adalimumab), methods of manufacture of thecompositions, methods of their administration, and kits containing thesame.

BACKGROUND OF THE INVENTION

Tumor necrosis factor alpha (TNFα) is a naturally occurring mammaliancytokine produced by various cell types, including monocytes andmacrophages in response to endotoxin or other stimuli. TNFα is a majormediator of inflammatory, immunological, and pathophysiologicalreactions (Grell, M., et al. (1995) Cell, 83: 793-802).

Soluble TNFα is formed by the cleavage of a precursor transmembraneprotein (Kriegler, et al. (1988) Cell 53: 45-53), and the secreted 17kDa polypeptides assemble to soluble homotrimer complexes (Smith, et al.(1987), J. Biol. Chem. 262: 6951-6954; for reviews of TNF, see Butler,et al. (1986), Nature 320:584; Old (1986), Science 230: 630). Thesecomplexes then bind to receptors found on a variety of cells. Bindingproduces an array of pro-inflammatory effects, including (i) release ofother pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, andIL-1, (ii) release of matrix metalloproteinases and (iii) up-regulationof the expression of endothelial adhesion molecules, further amplifyingthe inflammatory and immune cascade by attracting leukocytes intoextravascular tissues.

There are many disorders associated with elevated levels of TNFα. Forexample, TNFα has been shown to be up-regulated in a number of humandiseases, including chronic diseases such as rheumatoid arthritis (RA),inflammatory bowel disorders, including Crohn's disease and ulcerativecolitis, sepsis, congestive heart failure, asthma bronchiale andmultiple sclerosis. TNFα is also referred to as a pro-inflammatorycytokine.

Physiologically, TNFα is also associated with protection from particularinfections (Cerami. et al. (1988), Immunol. Today 9:28). TNFα isreleased by macrophages that have been activated by lipopolysaccharidesof Gram-negative bacteria. As such, TNFα appears to be an endogenousmediator of central importance involved in the development andpathogenesis of endotoxic shock associated with bacterial sepsis.

Adalimumab (Humira®, AbbVie, Inc.) is a recombinant human IgG1monoclonal antibody specific for human TNF. This antibody is also knownas D2E7. Adalimumab consists of 1330 amino acids and has a molecularweight of approximately 148 kilodaltons. Adalimumab has been describedand claimed in U.S. Pat. No. 6,090,382, the disclosure of which ishereby incorporated by reference in its entirety. Adalimumab is usuallyproduced by recombinant DNA technology in a mammalian cell expressionsystem, such as, for example, Chinese Hamster Ovary cells. Adalimumabbinds specifically to TNFα and neutralizes the biological function ofTNF by blocking its interaction with the p55 and p75 cell surface TNFreceptors.

Various formulations of adalimumab are known in the art. See, forexample, U.S. Pat. Nos. 8,216,583 and 8,420,081. There is still need forstable liquid formulations of adalimumab that allow its long termstorage without substantial loss in efficacy.

SUMMARY OF THE INVENTION

The invention provides stable aqueous formulations comprising adalimumabthat allow its long term storage.

In a first embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab; a stabilizercomprising at least one member selected from the group consisting of apolyol and a surfactant; and a buffer selected from the group consistingof citrate, phosphate, succinate, histidine, tartrate and maleate,wherein said composition has a pH of about 4 to about 8 and preferablyabout 5 to about 6, and wherein said buffer does not comprise acombination of citrate and phosphate, and preferably does not compriseany citrate buffer. In this embodiment, the stabilizer preferablycomprises both polyol and surfactant.

In a second embodiment, utilizing a single buffer system, the inventionprovides a stable aqueous pharmaceutical composition comprisingadalimumab, a polyol, a surfactant, and a buffer system comprising asingle buffering agent, said single buffering agent being selected fromcitrate, phosphate, succinate, histidine, tartrate or maleate, but notincluding combinations of the foregoing; wherein the formulation has apH of about 4 to 8, and preferably about 5 to about 6. Histidine andsuccinate are particularly preferred for use as single buffering agents.As used herein the term buffer, buffer system, or buffering agent, andlike terminology, is intended to denoted buffer components thatintroduce buffer capacity in the formulation in addition to anybuffering capacity offered by the protein itself, hence the term“buffer”, etc, is not intended to include the protein itself as a selfbuffering entity.

In a third embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab, a stabilizercomprising at least one member selected from a polyol and a surfactant,wherein said composition has a pH of about 4 to about 8, and preferablyabout 5 to about 6, and wherein said composition is substantially freeof a buffer.

In a fourth embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab, a polyol, and a bufferselected from the group consisting of citrate, phosphate, succinate,histidine, tartrate and maleate, wherein said composition has a pH ofabout 4 to about 8 and preferably about 5 to about 6, and wherein saidcomposition is free or substantially free of a surfactant. Preferably,the composition (i) does not contain the buffer combination of citrateand phosphate; and (ii) the buffer is at least one member selected fromthe group consisting of histidine and succinate; and (iii) the polyol isselected from the group consisting of mannitol, sorbitol and trehalose.

In a fifth embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab, a surfactant, and abuffer selected from the group consisting of citrate, phosphate,succinate, histidine, tartrate and maleate, wherein said composition hasa pH of about 4 to 8 and preferably about 5 to about 6, and wherein saidcomposition is substantially free of polyol. Preferably, the composition(i) does not contain the buffer combination of citrate and phosphate;and (ii) the buffer is at least one member selected from the groupconsisting of histidine and succinate, including combinations thereof.

In each of the five embodiments discussed above, the composition mayoptionally further comprise a stabilizer selected from the groupconsisting of an amino acid, a salt, ethylenediaminetetraacetic acid(EDTA) and a metal ion. The amino acid stabilizer may be selected fromthe group consisting of glycine, alanine, glutamate, arginine andmethionine. The salt stabilizer may be selected from the groupconsisting of sodium chloride and sodium sulfate. The metal ionstabilizer may be selected from the group consisting of zinc, magnesiumand calcium. Preferably, adalimumab formulations containing thestabilizers mentioned above also do not contain buffer systems in whichphosphate buffer and citrate buffer are present in combination, and,most preferably contains buffer systems free or substantially free ofcitrate buffer. In particularly preferred embodiments, (i) the optionaladditional stabilizer present in this embodiment is not sodium chloride,or comprises sodium chloride present in amounts not to exceed about 100mM, and comprises at least one of arginine and glycine, includingcombinations of the two amino acids; (ii) the buffer, when present,contains no citrate, or at least no citrate and phosphate combination,but is instead at least one of histidine and succinate, includingcombinations thereof; and (iii) the stabilizer when it includes a polyolis preferably mannitol in amounts exceeding about 150 mM.

In further embodiments the invention is directed to an aqueous, bufferedpharmaceutical composition comprising adalimumab and a buffer, wherein(i) the composition is free or substantially free of a buffercombination that comprises both a citrate buffer and a phosphate buffer;and (ii) the composition exhibits long term stability.

Another embodiment of the invention concerns an aqueous, bufferedpharmaceutical composition exhibiting long term stability, saidcomposition comprising: (i) adalimumab; (ii) a buffer selected from thegroup consisting of histidine buffer, succinate buffer, and combinationsthereof; (iii) a polysorbate or poloxamer surfactant, or combinationsthereof; and (iv) one or both of the following: (a) a stabilizerselected from the group consisting of glycine, alanine, glutamate,arginine, methionine, EDTA, sodium chloride, sodium sulfate, metal ions,and combinations thereof; and (b) a polyol selected from sorbitol,mannitol, and trehalose, or combinations thereof. Optionally, theformulation may also include a sugar, such as sucrose.

In a further embodiment the invention is an aqueous, bufferedpharmaceutical composition comprising adalimumab and a buffer, wherein(i) the composition is free or substantially free of a polyol; and (ii)the composition exhibits long term stability.

In still a further embodiment the invention is directed to an aqueous,buffered pharmaceutical composition comprising adalimumab and a buffer,wherein (i) the composition is free or substantially free of surfactant;and (ii) the composition exhibits long term stability.

Another embodiment of the inventions concerns an aqueous pharmaceuticalcomposition comprising adalimumab wherein: (i) the composition is freeor substantially free of buffer; and (ii) the composition exhibits longterm stability.

In another embodiment, the adalimumab formulation of the presentinvention comprises, consists of, or consists essentially of,adalimumab, histidine buffer as the sole buffer in the formulation,glycine (or arginine, or combinations thereof) as the sole stabilizeramong the non-surfactant stabilizers referenced earlier, and polysorbate80. In this formulation, the amount of adalimumab is 20 to 150 mg/ml,the amount of histidine buffer is up to about 50 mM, the amount ofglycine is up to about 300 mM, and the amount of polysorbate 80 is inthe range of about 0.01 to about 0.2 wt %. Optionally, this formulationmay include up to about 100 mM NaCl. The present invention alsocontemplates modification of this formulation to combine the histidinebuffer with one or more of citrate, acetate, phosphate, maleate,tartrate buffers.

In yet another embodiment, the adalimumab formulation of the presentinvention comprises, consists of, or consists essentially of,adalimumab, histidine buffer as the sole buffer, mannitol (or sorbitolor trehelose), and polysorbate 80, and further being free orsubstantially free of the non-surfactant stabilizers (e.g. glycine,arginine, etc.) referenced above. In this formulation, the amount ofadalimumab is 20 to 150 mg/ml, the amount of histidine buffer is up toabout 50 mM; the amount of polyol is up to about 300 mM; and the amountof polysorbate 80 is in the range of about 0.01 to about 0.2 wt %.Optionally, this formulation may include up to about 100 mM NaCl. Thepresent invention also contemplates modification of this formulation tocombine the histidine buffer with one or more of citrate, acetate,phosphate, maleate, tartrate buffers.

In a method aspect, the invention is directed to a method for enhancinglong term stability in an aqueous, buffered adalimumab formulation,comprising one or more of the steps of: (a) incorporating histidinebuffer, succinate buffer, or a combination thereof, in the formulationbased on empirical data indicating that such buffers contribute to thestability of the formulation to a greater extent than other buffers orbuffer combinations; or (b) incorporating glycine, arginine or acombination thereof as stabilizers in the formulation, based uponempirical data indicating that such stabilizer contribute to thestability of the formulation to a greater extent than other stabilizers;or (c) substantially excluding the presence of buffer or buffercombinations comprising citrate buffer (especially buffer combinationscomprising both citrate and phosphate) based upon empirical dataindicating that such buffer or buffer combinations perform poorly interms of stabilizing the formulation in comparison to other buffers. Themethod may further comprise the selection of PS 80 as a surfactant basedon empirical data indicating that PS 80 imparts better thermal stabilityto the adalimumab formulation than other surfactants, including PS 20.The method is useful to obtain a formulation of adalimumab that exhibitslong term stability comparable to or better than commercially availableadalimumab formulations marketed under the trademark Humira®.

In a further method aspect, the invention is directed to a method fortreating an inflammatory condition in a subject which comprisesadministering to such subject any of the adalimumab formulationembodiments as described herein.

In the foregoing embodiments, where the above referenced stabilizers maybe included in the formulations, it is further discovered thatsatisfactory stabilization can be attained when such stabilizers areused in place of both polyol and surfactant and hence stabilizedformulations of the present invention can be free or substantially freeof both polyol and surfactant. Accordingly, in a sixth embodiment, theinvention provides a stable aqueous pharmaceutical compositioncomprising adalimumab, optionally a buffer, a stabilizer selected fromthe group consisting of an amino acid, a salt, EDTA, and a metal ion,and wherein said composition has a pH of about 4 to about 8, andpreferably 5 to about 6, and wherein said composition is eithersubstantially free of both polyol and surfactant. When buffer is presentin this embodiment, it is especially preferred that (i) the buffer notinclude the combination of citrate and phosphate; (ii) the buffer isselected from the group consisting of histidine and succinate; and (iii)the stabilizer does not comprise sodium chloride, but instead is atleast one member selected from the group consisting of arginine andglycine.

Important aspects of the present invention in certain embodimentsinclude (i) that sorbitol and trehalose are discovered to besignificantly better stabilizers of adalimumab formulations thanmannitol, unless mannitol is used at concentrations in excess of about200-300 mM in which case the three are generally equivalent; (ii)arginine and glycine (and combinations) are discovered to besignificantly better stabilizers of adalimumab formulations than sodiumchloride; and; (iii) when buffers are used in the formulation, it isdiscovered that the combination of citrate and phosphate is surprisinglysignificantly poorer in stabilizing adalimumab than other buffers suchas succinate, histidine, phosphate and tartrate. The relatively poorperformance of the buffer combination of citrate and phosphate is ratherunexpected considering the apparent importance attributed to the use ofa citrate/phosphate combined buffer in U.S. Pat. No. 8,216,583. To thecontrary, we have now found that a phosphate/citrate buffer combinationis not an optimal choice for obtaining a stabilized adalimumabformulation, and in fact, an element of our invention is the discoverythat this combination should be avoided altogether in favor of otherbuffer systems.

Preferably, a polyol is a sugar alcohol; and even more preferably, thesugar alcohol is selected from the group consisting of mannitol,sorbitol and trehalose. However, as between mannitol and sorbitol, theinvention has discovered, as noted above, a distinct stabilizationadvantage in using sorbitol or trehalose instead of mannitol, unlessmannitol is used at concentrations in excess of about 200 mM, in whichcase mannitol, sorbitol and trehalose are generally equivalent. Atconcentrations below about 200 mM, mannitol has been found to be apoorer stabilizer than sorbitol or trehalose in an adalimumabformulation.

Preferably, a surfactant is a polysorbate or poloxamer; and even morepreferably PS 80, PS 40, PS20, Pluronic F-68 and combinations. We havediscovered a distinct and surprising thermal stabilization advantage inselecting PS 80 instead of PS-20.

These and other aspects will become apparent from the followingdescription of the various embodiments, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Furtherrepresentative embodiments are set forth in the numerous formulationstudies reported in the detailed description, as well as the variousembodiments listed in Appendices A, B and C attached hereto and made apart hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a bar chart of stability of various adalimumab formulations asdetermined by size exclusion chromatography (SEC).

FIG. 2 is a bar chart of stability of various adalimumab formulations asdetermined by reversed phase (RP) high performance liquid chromatography(HPLC).

FIG. 3 is a graph of a partial least squares (PLS) model 1 demonstratingeffect of citrate/phosphate on stability.

FIG. 4 is a graph of a PLS model 2 demonstrating effect ofcitrate/phosphate on stability.

FIG. 5 is a graph of a PLS model 1 demonstrating effect ofhistidine/glycine on stability.

FIG. 6 is a graph of a PLS model 1 demonstrating effect ofarginine/sorbitol on stability.

FIG. 7 is a graph of a PLS model 1 demonstrating effect of pH/histidineon stability.

FIG. 8 is a graph of a PLS model 2 demonstrating effect of pH/histidineon stability.

FIG. 9 is a graph of a PLS model 2 demonstrating effect oftrehalose/PS80 on stability.

FIG. 10 is a graph of a PLS model 2 demonstrating effect ofmannitol/PS80 on stability.

FIG. 11 is a graph of a PLS model 1 demonstrating effect ofmannitol/NaCl on stability.

FIG. 12 is a graph of a PLS model 1 demonstrating effect ofEDTA/methionine on stability.

FIG. 13 is a graph of a PLS model A demonstrating effect of citrate andphosphate on stability.

FIG. 14 is a graph of a PLS model A demonstrating effect of pH andhistidine buffer on stability.

FIG. 15 is a graph of a PLS model A demonstrating effect of glycine andarginine on stability.

FIG. 16 is a graph of a PLS model A demonstrating effect of NaCl andpolysorbate 80 (PS 80) on stability.

FIG. 17 is a graph of a PLS model B demonstrating effect of citrate andphosphate on stability.

FIG. 18 is a graph of a PLS model B demonstrating effect of pH andhistidine buffer on stability.

FIG. 19 is a graph of a PLS model B demonstrating effect arginine andglycine on stability.

FIG. 20 is a graph of a PLS model B demonstrating effect of PS80 andmannitol on stability.

FIG. 21 is a graph of a PLS model B demonstrating effect of EDTA andNaCl on stability.

FIG. 22 is a graph of a PLS model B demonstrating effect of succinatebuffer and histidine buffer on stability.

FIG. 23 is a graph of a PLS model C demonstrating effect of citrate andphosphate on stability.

FIG. 24 is a graph of a PLS model C demonstrating effect of pH andhistidine buffer on stability.

FIG. 25 is a graph of a PLS model C demonstrating effect of arginine andglycine on stability.

FIG. 26 is a graph of a PLS model C demonstrating effect of mannitol andPS 80 on stability.

FIG. 27 is a graph of a PLS model C demonstrating effect of PS 80 andNaCl on stability.

FIG. 28 is a graph of a PLS model C demonstrating effect of pH andprotein concentration on stability.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail. Asused in the description and throughout the claims, the meaning of “a”,“an”, and “the” includes plural reference unless the context clearlydictates otherwise. Also, as used in the description and throughout theclaims, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Additionally, some terms used in thisspecification are more specifically defined below.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

“Around,” “about” or “approximately” shall generally mean within 20percent, within 10 percent, within 5, 4, 3, 2 or 1 percent of a givenvalue or range. Numerical quantities given are approximate, meaning thatthe term “around,” “about” or “approximately” can be inferred if notexpressly stated.

The term “adalimumab” is synonymous with the active pharmaceuticalingredient in Humira® as well as protein considered or intended asbiosimilar or bio-better variants thereof. Adalimumab is a recombinanthuman IgG1 monoclonal antibody specific for human TNF. Adalimumab isalso known as D2E7. Adalimumab has two light chains, each with amolecular weight of approximately 24 kilodaltons (kDa) and two IgG1heavy chains each with a molecular weight of approximately 49 kDa. Eachlight chain consists of 214 amino acid residues and each heavy chainconsists of 451 amino acid residues. Thus, adalimumab consists of 1330amino acids and has a total molecular weight of approximately 148 kDa.The term adalimumab is also intended to encompass so-called bio-similaror bio-better variants of the adalimumab protein used in commerciallyavailable Humira®. For example, a variant of commercial Humira® may beacceptable to the FDA when it has essentially the same pharmacologicaleffects as commercially available Humira®, even though it may exhibitcertain physical properties, such as glycosylation profile, that may besimilar if not identical to Humira®.

For the purposes of the present application, the term “adalimumab” alsoencompasses adalimumab with minor modifications in the amino acidstructure (including deletions, additions, and/or substitutions of aminoacids) or in the glycosylation properties, which do not significantlyaffect the function of the polypeptide. The term “adalimumab”encompasses all forms and formulations of Humira®, including but notlimited to concentrated formulations, injectable ready-to-useformulations; formulations reconstituted with water, alcohol, and/orother ingredients, and others.

The term “human TNFα” (which may be abbreviated as hTNFα, or simplyhTNF), as used herein, is intended to refer to a human cytokine thatexists as a 17 kD secreted form and a 26 kD membrane associated form,the biologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.).

The term “antibody”, as used herein, refers to immunoglobulin moleculescomprised of four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor VH) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH1, CH2 and CH3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In one embodiment of the invention, the formulation containsan antibody with CDR1, CDR2, and CDR3 sequences like those described inU.S. Pat. Nos. 6,090,382; 6,258,562, and 8,216,583.

An antibody or antigen-binding portion thereof may be part of a largerimmunoadhesion molecule, formed by covalent or noncovalent associationof the antibody or antibody portion with one or more other proteins orpeptides. Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such asFab and F(ab′)₂ fragments, can be prepared from whole antibodies usingconventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein.

The term “isolated antibody”, as used herein, refers to an antibody thatis substantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds hTNFαis substantially free of antibodies that specifically bind antigensother than hTNFα). An isolated antibody that specifically binds hTNFαmay, however, have cross-reactivity to other antigens, such as TNFαmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “glycine” refers to an amino acid whose codons are GGT, GGC,GGA, and GGG.

The tern “arginine” refers to an α-amino acid whose codons are CCU, CCC,CCA, and CCG.

The term “alanine” refers to an amino acid whose codons are GCT, GCC,GCA, and GCG.

The term “methionine” refers to an amino acid whose codon is ATG.

The term “glutamate” refers to an amino acid whose codons are GAA andGAG.

The term “sugar” refers to monosaccharides, disachharides, andpolysaccharides. Examples of sugars include, but are not limited to,sucrose, glucose, dextrose, and others.

The term “polyol” refers to an alcohol containing multiple hydroxylgroups. Examples of polyols include, but are not limited to, mannitol,sorbitol, and others.

The term “metal ion” refers to a metal atom with a net positive ornegative electric charge. For the purposes of the present application,the term “metal ion” also includes sources of metal ions, including butnot limited to metal salts.

The term “long-term storage” or “long term stability” is understood tomean that the pharmaceutical composition can be stored for three monthsor more, for six months or more, and preferably for one year or more,most preferably a minimum stable shelf life of at least two years.Generally speaking, the terms “long term storage” and “long termstability” further include stable storage durations that are at leastcomparable to or better that the stable shelf typically required forcurrently available commercial formulations of adalimumab, withoutlosses in stability that would render the formulation unsuitable for itsintended pharmaceutical application. Long-term storage is alsounderstood to mean that the pharmaceutical composition is stored eitheras a liquid at 2-8° C., or is frozen, e.g., at −20° C., or colder. It isalso contemplated that the composition can be frozen and thawed morethan once.

The term “stable” with respect to long-term storage is understood tomean that adalimumab contained in the pharmaceutical compositions doesnot lose more than 20%, or more preferably 15%, or even more preferably10%, and most preferably 5% of its activity relative to activity of thecomposition at the beginning of storage.

The term “substantially free” means that either no substance is presentor only minimal, trace amounts of the substance are present which do nothave any substantial impact on the properties of the composition. Ifreference is made to no amount of a substance, it should be understoodas “no detectable amount”.

The term “mammal” includes, but is not limited to, a human.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “composition” refers to a mixture that usually contains acarrier, such as a pharmaceutically acceptable carrier or excipient thatis conventional in the art and which is suitable for administration intoa subject for therapeutic, diagnostic, or prophylactic purposes. It mayinclude a cell culture in which the polypeptide or polynucleotide ispresent in the cells or in the culture medium. For example, compositionsfor oral administration can form solutions, suspensions, tablets, pills,capsules, sustained release formulations, oral rinses or powders.

The terms “pharmaceutical composition” and “formulation” are usedinterchangeably.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease, for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function; or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect,covering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse affect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “disease” refers to any condition, infection, disorder orsyndrome that requires medical intervention or for which medicalintervention is desirable. Such medical intervention can includetreatment, diagnosis and/or prevention.

The term “therapeutically effective amount” refers to an amount which,when administered to a living subject, achieves a desired effect on theliving subject. For example, an effective amount of the polypeptide ofthe invention for administration to the living subject is an amount thatprevents and/or treats an integrin αvβ3-mediated disease. The exactamount will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for systemic versus localized delivery,age, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

EMBODIMENTS OF THE INVENTION

When pharmaceutical compositions containing adalimumab (Humira®),including aqueous and lyophilized formulations of adalimumab are storedon a long-term basis, the activity of adalimumab can be lost ordecreased due to aggregation and/or degradation. Thus, the presentinvention provides aqueous formulations of adalimumab that allow stablelong-term storage of adalimumab, so that adalimumab is stable over thecourse of storage either in liquid or frozen states. The providedformulations do not require any extra steps such as rehydrating.

Numerous embodiments of the present invention are explained in a greaterdetail below.

Adalimumab

All of the compositions of the present invention comprise adalimumab. Asexplained in the Background section of this application, adalimumab is arecombinant human IgG1 monoclonal antibody specific for human tumornecrosis factor (TNF). This antibody is also known as D2E7. Adalimumabconsists of 1330 amino acids and has a molecular weight of approximately148 kilodaltons. Adalimumab has been described and claimed in U.S. Pat.No. 6,090,382. The term “adalimumab” is also intended to mean so-called“bio-similar” and “bio-better” versions of the active adalimumab proteinpresent in commercially available Humira®.

Adalimumab suitable for storage in the present pharmaceuticalcomposition can be produced by standard methods known in the art. Forexample, U.S. Pat. Nos. 6,090,382 and 8,216,583 describe various methodsthat a skilled artisan could use to prepare adalimumab protein for usein the formulations of the present invention. These methods areincorporated by reference herein. For example, adalimumab can beprepared by recombinant expression of immunoglobulin light and heavychain genes in a host cell.

Purification of the expressed adalimumab can be performed by anystandard method. When adalimumab is produced intracellularly, theparticulate debris is removed, for example, by centrifugation orultrafiltration. When adalimumab is secreted into the medium,supernatants from such expression systems can be first concentratedusing standard polypeptide concentration filters. Protease inhibitorscan also be added to inhibit proteolysis and antibiotics can be includedto prevent the growth of microorganisms.

Adalimumab can be purified using, for example, hydroxyapatitechromatography, gel electrophoresis, dialysis, and affinitychromatography, and any combination of known or yet to be discoveredpurification techniques, including but not limited to Protein Achromatography, fractionation on an ion-exchange column, ethanolprecipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSET®, an anion or cation exchange resinchromatography (such as a polyaspartic acid column), chromatofocusing,SDS-PAGE, and ammonium sulfate precipitation.

I Formulations of Adalimumab with a Polyol and/or Surfactant, butwithout a Citrate/Phosphate Buffer

In a first embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab; a stabilizercomprising at least one member selected from the group consisting of apolyol and a surfactant; and a buffer selected from the group consistingof citrate, phosphate, succinate, histidine, tartrate and maleate,wherein said composition has a pH of about 4 to about 8, and preferablyabout 5 to about 6, and wherein said buffer does not comprise acombination of citrate and phosphate. In this embodiment, the stabilizerpreferably comprises both polyol and surfactant. The pharmaceuticalcomposition can comprise one, or any combination of two or more buffers,as long as it does not comprise both citrate and phosphate. Thesurfactant may be any pharmaceutically acceptable surfactant, preferablypolysorbates (e.g., polysorbate 80) or poloxamers (e.g., Pluronic F-68).

II Formulations of Adalimumab Using a Simile Buffer System

In a second embodiment, utilizing a single buffer system, the inventionprovides a stable aqueous pharmaceutical composition comprisingadalimumab, a polyol, a surfactant, and a buffer system comprising asingle buffering agent, said single buffering agent being selected fromcitrate, phosphate, succinate, histidine, tartrate or maleate, but notincluding combinations of the foregoing; wherein the formulation has apH of about 4 to 8, and preferably about 5 to about 6. Histidine andsuccinate are particularly preferred for use as single buffering agents.It was surprisingly discovered that adalimumab compositions whichcomprise only one buffer (as opposed to two or more buffers) are morestable than adalimumab compositions comprising both a citrate buffer anda phosphate buffer. In the single buffer embodiment, adalimumab can bepresent at a concentration from about 20 to about 150 mg/ml, morepreferably from about 20 to about 100 mg/ml, and even more preferablyfrom about 30 to about 50 mg/ml. The buffer is present at aconcentration from about 5 mM to about 50 mM. The pH of the compositionsis between about 5 and about 6. The single buffer compositions of theinvention may further comprise a stabilizer selected from the groupconsisting of an amino acid, a salt, ethylenediaminetetraacetic acid(EDTA) and a metal ion. The amino acid is selected from the groupconsisting of glycine, alanine, glutamate, arginine and methionine, mostpreferably glycine, arginine and methionine. The salt is selected fromthe group consisting of sodium chloride and sodium sulfate. The metalion is selected from the group consisting of zinc, magnesium andcalcium. The compositions of the invention may further comprise asurfactant. The surfactant is a polysorbate surfactant or a poloxamersurfactant. Polysorbate surfactants include polysorbate 80, polysorbate40 and polysorbate 20. A preferred polysorbate surfactant is polysorbate80. Poloxamer surfactants include poloxamer 188 (also availablecommercially as Pluronic F-68). Most preferably, the surfactant ispolysorbate 80. The single buffer composition may further comprise apolyol. Preferably, the polyol is a sugar alcohol; and even morepreferably, the sugar alcohol is mannitol, sorbitol or trehalose. Thesingle buffer adalimumab composition may also comprise a sugar,preferably sucrose, glucose or dextrose.

In one embodiment of a single buffer adalimumab formulation, theinvention provides a stable aqueous pharmaceutical compositioncomprising adalimumab at a concentration from about 20 and about 150mg/ml, polysorbate 80 at a concentration from about 1 to 50 μM, andsuccinate at a concentration from about 5 mM and about 50 mM, whereinsaid composition has a pH of about 5 to about 5.5, and wherein saidcomposition is substantially free of any other buffers.

In another embodiment of a single buffer adalimumab formulation, theinvention provides a stable aqueous pharmaceutical compositioncomprising adalimumab at a concentration from about 20 and about 150mg/ml, polysorbate 80 at a concentration from about 1 to 50 μM, andhistidine at a concentration from about 5 mM and about 50 mM, whereinsaid composition has a pH of about 5 to about 5.5, and wherein saidcomposition is substantially free of any other buffers.

In a further embodiment of a single buffer adalimumab formulation, theinvention provides a stable aqueous pharmaceutical compositioncomprising adalimumab at a concentration from about 20 and about 150mg/ml, polysorbate 80 at a concentration from about 1 to 50 μM, andeither tartrate, maleate or acetate at a concentration from about 5 mMand about 50 mM, wherein said composition has a pH of about 5 to about5.5, and wherein said composition is substantially free of any otherbuffers.

III Formulations of Adalimumab which Exclude Buffer

In a third embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab, a stabilizercomprising at least one member selected from a polyol and a surfactant,wherein said composition has a pH of about 4 to about 8 and preferablyabout 5 to about 6, and wherein said composition is substantially freeof a buffer. The term “free of buffer” should be understood to allowinclusion of the inherent buffering effect of the protein itself. In abuffer free formulation, the stabilizers referenced above may also bepresent (e.g. glycine, arginine and combinations thereof).

IV Formulations of Adalimumab which Exclude Surfactant

In a fourth embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab, a polyol, and a bufferselected from the group consisting of citrate, phosphate, succinate,histidine, tartrate and maleate, wherein said composition has a pH ofabout 4 to about 8 and preferably about 5 to about 6, and wherein saidcomposition is free or substantially free of a surfactant. Preferably,the composition (i) does not contain the buffer combination of citrateand phosphate; and (ii) the buffer is at least one member selected fromthe group consisting of histidine and succinate; and (iii) the polyol isnot mannitol at concentrations less than about 150 mM, but instead isselected from the group consisting of mannitol at concentrationsexceeding about 150 mM, sorbitol and trehalose.

V Formulations of Adalimumab which Exclude Polyol

In a fifth embodiment, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab, a surfactant, and abuffer selected from the group consisting of citrate, phosphate,succinate, histidine, tartrate and maleate, wherein said composition hasa pH of about 4 to about 8, and preferably about 5 to about 6, andwherein said composition is substantially free of polyol. Preferably,the composition (i) does not contain the buffer combination of citrateand phosphate; and (ii) the buffer is at least one member selected fromthe group consisting of histidine and succinate.

Additional Stabilizers Useful in Embodiments I Through V.

Optionally, in each of the five embodiments summarized above, thecomposition may further comprise a stabilizer selected from the groupconsisting of an amino acid, a salt, ethylenediaminetetraacetic acid(EDTA) and a metal ion. The amino acid stabilizer may be selected fromthe group consisting of glycine, alanine, glutamate, arginine andmethionine. The salt stabilizer may be selected from the groupconsisting of sodium chloride and sodium sulfate. The metal ionstabilizer may be selected from the group consisting of zinc, magnesiumand calcium. Preferably, adalimumab formulations containing thestabilizers mentioned above also do not contain buffer systems in whichphosphate buffer and citrate buffer are present in combination. Mostpreferably (i) the optional additional stabilizer present in thisembodiment is not sodium chloride, and comprises at least one or both ofarginine and glycine; (ii) the buffer, when present, contains no citrateand phosphate combination but is instead at least one of histidine andsuccinate; and (iii) the stabilizer when it includes a polyol is notmannitol unless in amounts greater than about 150 mM, and may alsoinclude trehalose and sorbitol. Preferably the amount of mannitol isgreater than about 150 mM, and most preferably greater than about 200mM.

VI Formulations of Adalimumab Replacing Both Surfactant and Polyol withOther Stabilizers

It has been further discovered that satisfactory stabilization can beattained when the stabilizers mentioned above are used in place of bothpolyol and surfactant, accordingly, in a sixth embodiment, the inventionprovides a stable aqueous pharmaceutical composition comprisingadalimumab, optionally a buffer, a stabilizer selected from the groupconsisting of an amino acid, a salt, EDTA, and a metal ion, and whereinsaid composition has a pH of about 4 to about 8, and preferably about 5to about 6, and wherein said composition is free or substantially freeof a polyol and surfactant. When buffer is present in this embodiment,it is especially preferred that (i) the buffer not include thecombination of citrate and phosphate; (ii) the buffer is selected fromthe group consisting of histidine and succinate; and (iii) thestabilizer does not comprise sodium chloride, but instead is at leastone member selected from the group consisting of arginine and glycine.It is also preferred that the buffer is free or substantially free ofcitrate buffer, as we have discovered that it is generally poorer interms of stability contribution than other buffers, such as histidineand succinate.

In each of the embodiments above at least one of the followingadvantageous conditions can be optionally present (unless stated asbeing required): (i) the buffer preferably does not contain acombination of citrate and phosphate, or is free or substantially freeof citrate buffer (ii) the buffer preferably is at least one memberselected from the group consisting of histidine and succinate; and (iii)the stabilizer preferably does not include sodium chloride, or ifpresent is controlled to levels less than about 100 mM; (iv) thestabilizer is at least one member selected from the group consisting ofarginine and glycine, including combinations thereof; and (v) the polyolis preferably not mannitol (unless mannitol is present in amountsgreater than about 150 mM and preferably greater than about 200 mM) butmay include sorbitol and trehalose. When using polyols forstabilization, mannitol is discovered herein to be destabilizing incomparison to sorbitol and trehalose unless the mannitol is present inamounts generally above about 150 to 200 mM. When using otherstabilizers, it is discovered herein that sodium chloride isdestabilizing compared to arginine or glycine, but we observe somestabilization when the levels of sodium chloride are controlled to lessthan about 100 mM and preferably less than about 75 mM.

Preferably, adalimumab is present in the composition of the presentinvention at a concentration from about 20 to about 150 mg/ml, morepreferably from about 20 to about 100 mg/ml, and even more preferablyfrom about 30 to about 50 mg/ml.

Buffer, if present, is present at a concentration from about 5 mM toabout 50 mM.

Surfactant, if present, is preferably a polysorbate (PS). In an evenmore preferred embodiment, the polysorbate is polysorbate 80 (PS 80).Poloxamer surfactants are also suitable (e.g., Pluronic® F-68).

The polyol, if present, is a sugar alcohol. In an even more preferredembodiment, the sugar alcohol is selected from the group consisting ofmannitol, sorbitol and trehalose, and most preferably sorbitol andtrehalose.

Preferably, the polyol is at a concentration from about 1 to about 10%,more preferably, from about 2 to about 6%, and even more preferably fromabout 3 to 5%, wherein said values are weight by volume (w/v) of thetotal composition.

A stabilizer, when present, can be selected from the group consisting ofan amino acid, a salt, ethylenediaminetetraacetic acid (EDTA) and ametal ion. The amino acid can be selected from the group consisting ofglycine, alanine, glutamate, arginine and methionine. The salt may beselected from the group consisting of sodium chloride and sodiumsulfate. The metal ion may be selected from the group consisting ofzinc, magnesium and calcium. Glycine and arginine are particularlypreferred stabilizers.

Zinc, magnesium and calcium, when present for stabilization, may be at aconcentration from about 1 mM to about 100 mM, and more preferably fromabout 1 to about 10 mM.

Glycine, or arginine, or combinations thereof, if present forstabilization, is at a total concentration of up to about 300 mM, andpreferably about 150 to 300 mM.

Methionine, if present for stabilization, is present at a concentrationfrom about 1 to about 10 mg/ml, more preferably from about 1 mg/ml toabout 5 mg/ml.

Sodium chloride, if present for stabilization, is at a concentrationfrom about 5 to about 150 mM, more preferably, from about 20 to about140 mM, and even more preferably less than about 100 mM.

Sodium sulfate, if present if present for stabilization, is at aconcentration from about 5 to about 150 mM, more preferably, from about20 to about 120 mM, and even more preferably from about 60 to about 100mM.

EDTA, if present for stabilization, is present at a concentration fromabout 0.01% to about 0.05%, more preferably from about 0.05% to about0.25%, and even more preferably from about 0.08% to about 0.2%.

Preferably, the pH of the composition is from about 5 to about 5.5; andeven more preferably is about 5.2 to 5.4.

In an example of Embodiment I and II, above, the invention provides astable aqueous pharmaceutical composition comprising adalimumab at aconcentration from about 20 and about 150 mg/ml, sorbitol or trehaloseat a concentration from about 1 to 10% weight by volume, polysorbate 80at a concentration from about 1 to 50 μM, and at least one of succinate,histidine, phosphate, tartrate, maleate or citrate buffer, at aconcentration from about 5 mM to about 50 mM, wherein said compositionhas a pH of about 5 to about 5.5, and provided said composition is freeor substantially free of citrate/phosphate buffer combination. Further,we rank citrate as the poorest of buffers, and preferably avoid italthough it is still within the scope of the invention to formulatestable formulations of adalimumab that include citrate buffer, if notthe combination thereof with phosphate.

In an example of Embodiment IV, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab at a concentration fromabout 20 and about 150 mg/ml, sorbitol or trehalose at a concentrationfrom about 1 to 10% weight by volume, and at least one of succinate,histidine, phosphate, tartrate, maleate or citrate buffer, at aconcentration from about 5 mM to about 50 mM, wherein said compositionhas a pH of about 5 to about 5.5, and wherein said composition issubstantially free of a surfactant and, optionally, and preferably, freeor substantially free of citrate/phosphate buffer combination.

In an example of Embodiment VI, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab at a concentration fromabout 20 and about 150 mg/ml, glycine at a concentration from about 20to about 200 mM, and at least one of succinate, histidine, phosphate,tartrate, maleate or citrate buffer, at a concentration from about 5 mMto about 50 mM, wherein said composition has a pH of about 5 to about5.5, and wherein said composition is free or substantially free polyol;surfactant (e.g. PS8) is preferably, but optionally present; and thecomposition is, optionally, and preferably, free or substantially freeof citrate/phosphate buffer combination.

In a further example of Embodiment VI, the invention provides a stableaqueous pharmaceutical composition comprising adalimumab at aconcentration from about 20 and about 150 mg/ml, arginine or glycine ata concentration from about 1 to about 250 mM, and at least one ofsuccinate, histidine, phosphate, tartrate, maleate or citrate buffer, ata concentration from about 5 mM and about 50 mM wherein said compositionhas a pH of about 5 to about 5.5, and wherein said composition issubstantially free of polyol. Surfactant (e.g. PS80) is preferably butoptionally present, and the composition is, optionally, and preferably,free or substantially free of citrate/phosphate buffer combination.

In a further example of Embodiment VI, the invention provides a stableaqueous pharmaceutical composition comprising adalimumab at aconcentration from about 20 and about 150 mg/ml, sodium chloride at aconcentration from about 5 to about 150 mM, and at least one ofsuccinate, histidine, phosphate, tartrate, maleate or citrate buffer, ata concentration from about 5 mM and about 50 mM wherein said compositionhas a pH of about 5 to about 5.5, and wherein said composition is freeor substantially free of a polyol. Surfactant (e.g. PS80) is preferablybut optionally present; and the composition is, optionally, andpreferably, free or substantially free of citrate/phosphate buffercombination.

In an example of Embodiment V, the invention provides a stable aqueouspharmaceutical composition comprising adalimumab at a concentration fromabout 20 and about 150 mg/ml, sodium chloride at a concentration fromabout 5 to about 150 mM, polysorbate 80 at a concentration from about 1to 50 μM, and at least one of succinate, histidine, phosphate, tartrate,maleate or citrate buffer, at a concentration from about 5 mM and about50 mM wherein said composition has a pH of about 5 to about 5.5, andwherein said composition is free or substantially free of a polyol and,optionally, and preferably, free or substantially free ofcitrate/phosphate buffer.

In an example of Embodiments I and II, with additional stabilization,the invention provides a stable aqueous pharmaceutical compositioncomprising adalimumab at a concentration from about 20 and about 150mg/ml, polysorbate 80 at a concentration from about 1 to about 50 μM,sorbitol or trehalose at a concentration from about 1 to about 10%weight by volume, EDTA at a concentration from about 0.01% to about0.5%, and at least one of succinate, histidine, phosphate, tartrate,maleate or citrate, as a sole buffer, at a concentration from about 5 mMand about 50 mM wherein said composition has a pH of about 5 to about5.5, and wherein the composition is free, or substantially free ofcitrate/phosphate buffer combination.

In a further example of Embodiments I and II, with additionalstabilization, the invention provides a stable aqueous pharmaceuticalcomposition comprising adalimumab at a concentration from about 20 andabout 150 mg/ml, polysorbate 80 at a concentration from about 1 to about50 μM, sorbitol or trehalose at a concentration from about 1 to about10% weight by volume, methionine at a concentration from about 1 toabout 10 mg/ml, %, and at least one of succinate, histidine, phosphate,tartrate, maleate or citrate at a concentration from about 5 mM andabout 50 mM wherein said composition has a pH of about 5 to about 5.5,wherein the composition is free or substantially free of anycitrate/phosphate buffer combination.

In a further example of Embodiments I and II, with additional amino acidstabilization, the invention provides a stable aqueous pharmaceuticalcomposition comprising adalimumab at a concentration from about 20 andabout 150 mg/ml, polysorbate 80 at a concentration from about 1 to about50 μM, mannitol, sorbitol or trehalose (preferably sorbitol) at aconcentration from about 1 to about 10% weight by volume, and amino acidthat is preferably one and not both of (a) arginine at a concentrationfrom about 1 to about 250 mg/ml, and (b) glycine at a concentration ofabout 20 to 200 mg/ml, and histidine buffer or succinate buffer at aconcentration from about 5 mM and about 50 mM, and wherein saidcomposition has a pH of about 5 to about 5.5; and wherein thecomposition is free or substantially free of any citrate/phosphatebuffer combination.

In a further example of Embodiment IV, with additional amino acidstabilization, the invention provides a stable aqueous pharmaceuticalcomposition comprising adalimumab at a concentration from about 20 andabout 150 mg/ml, polysorbate 80 at a concentration from about 1 to about50 μM, arginine at a concentration from about 1 to about 250 mg/ml,glycine at a concentration of about 20 to 200 mg/ml, and histidinebuffer or succinate buffer at a concentration from about 5 mM to about50 mM, and wherein said composition has a pH of about 5 to about 5.5 andis free or substantially free of polyol; and, optionally, wherein thecomposition is preferably free of any citrate/phosphate buffercombination.

Numerous embodiments of the adalimumab formulations of the presentinvention were prepared in eight separate blocks of experiments,referred to herein as “Block A” through “Block H.” Each block had 12 to16 different formulations that were exposed to accelerated storageconditions, 1 week at 40° C. and 2 weeks at 25° C. For each time pointthe chemical and physical stability of the adalimumab protein wasmeasured by SEC, RP, UV, pH, CE-IEF and CE-SDS.

Materials and Methods

1. Equipment Used in the Formulation Studies

Equipment Manufacture Model Serial Number Balance Sartorius CPA124S23350022 pH meter Denver Model 250 E25006B100 Instrument UV Cary Bio 100EL07103025 HPLC Dionex 3 Ultimate 3000 UPLC 8047439 HPLC Dionex 2Ultimate 3000, 8036991 UPLC Beckman Beckman P/ACE 455436 CE Agilent CEAgilent 3DCE 1600A 3546G00736 Rocker Labnet Orbit P4 8091840 Plate

2. Chemicals and Materials Used in the Formulation Studies

Chemical/Materials Producer Purity Lot Number: Citrate Mallinckrodt ACSH28475 Phosphate Fisher FCC 103372 Fisher ACS 113670 Succinate SpectrumReagent ZM0462 Histidine Spectrum USP XV0239 Spectrum USP ZG0216Tartrate Spectrum FCC 1BC0152 Maleate TCI  >99% 206-738-1 Mannitol BDHUSP 57910 Glycine Spectrum FCC YM3312 Spectrum FCC 1BJ0243 Fisher Tissue070082 Grade Arginine Spectrum USP 2AK0238 Spectrum USP 1CB0771 SodiumChloride Mallinckrodt ACS J52619 Macron USP 26434 Polysorbate 80Sigma-Aldrich Low 028K5309 Peroxide Sorbitol Spectrum NF 1AH0521Trehalose Spectrum N/A 1AE0739 Acetate Mallinckrodt FCC H31613 EDTASigma 98.5% 057K00071 Methionine Spectrum USP ZF0377 F-68 Sigma CellCulture 057K00331 Polysorbate 20 Spectrum NF 1AE0882 Sodium dodecylsulfate Fluka ACS 1344034 Tris base Fisher ACS S61374 2-mercaptoethanolFisher Electrophoresis 107667

Part Material/Reagents Number Supplier Slide-A-Lyzers 7K cutoff 66373Thermo Mini Dialysis Units 69550 Thermo Millex ®-GV 0.22 μM, FilterSLGV004SL Millipore 1 mL Vials 4500050375 SCHOTT cIEF Gel PolymerSolution 477497 Beckman Coulter pI Marker Kit A58481 Beckman CoulterPharmalyte 3-10 ampholyte 17-0456-01 GE Healthcare Fused silicacapillary (50 μm TSP050375 Polymicro i.d.) SDS-MW gel buffer A10663Beckman 10 kD internal standard A26487 Beckman

3. HPLC Columns Used in the Formulation Studies

Column Company Part # Lot Poroshell 300SB- Agilent 660750-906 USZW003083C8, 2.1 × 75 mm, 5um Poroshell 300SB- Agilent 660750-906 USZW003073 C8,2.1 × 75 mm, 5um ACQUITY UPLC BEH200 Waters 186005225 138123331 SEC, 1.7um Column, 4.6 × 150 mm ACQUITY UPLC BEH200 Waters 186005225 01471130951SEC, 1.7 um Column, 4.6 × 150 mm

Processing of Humira®.

Block A experiments used adalimumab present in commercially availableHumira®. Humira® material was dialyzed as follows: 100 μL of Humira® wasplaced into Mini Dialysis units with a 3.5 MWCO and dialyzed in 1 L offormulation buffer for 24 hours at 4 to 8° C. A few samples didexperience a small increase in volume due to the dialysis, but never toextent that the concentration of the polysorbate 80 dropped below theCMC (critical micelle concentration).

The protein concentration for each formulation was measured by UVabsorbance spectroscopy, using an calculated experimental molarabsorptivity based on reported concentration of Humira®, 50 mg/mL. For anumber of the formulations the protein concentration was adjusted byusing a spin concentrator. The sample was placed in the spinconcentrator and rotated at 14,000 RPM for 30 to 60 secs. The proteinconcentration was then checked with UV. After the targeted proteinconcentration around 50 mg/mL was reached the samples were filteredthrough a 0.22 μM sterile filter into sterile vials in a biosafety hood.The samples were then placed on stability at 40° C. for one and twoweeks.

Processing of a Proprietary Adalimumab Protein.

The formulation studies described herein used a proprietary adalimumabbiosimilar protein which did not contain polysorbate 80. The materialwas dialyzed using 7,000 MWCO Slide-A-Lyzers in different formulationbuffers for 24 hours at a temperature range between 4 to 8° C. Afterdialysis the protein concentration was measured by UV and sample pH wasmeasured. The target concentration of samples was 50±2.5 mg/mL, whichwas adjusted if the sample concentration fell out of the above range.Some of the samples did experience an increase in sample volume do todilution, requiring the concentration of the protein to increase. Forthese samples the protein concentration was increased by using spinconcentrators, usually at 14,000 rpm for 30 to 60 secs. The pH of anumber of samples were adjusted using 1M NaOH or 1M HCl to reach thetarget pH of 5.2.

After the targeted protein concentration and pH of the samples weredetermined to be within experimental parameters, the samples werefiltered through a 0.22 μM sterile filter into sterile vials in abiosafety hood. The samples were then placed on stability at 40° C. forone week and 25° C. for two weeks.

Freeze-Thaw Conditions:

The freeze thaw samples were prepared on the day of analysis to matchwith t=0. The samples were frozen at −80° C. between 3 to 7 minutes. Thefrozen sample was then thawed at room temperature until all the ice hadthawed. The freeze and thaw cycle was repeated 5 times for each sample.

Agitation Studies.

The samples were aggregated at 150 rpm for 24 hours at 4° C. on arockerplate. A control was prepared and placed next to the rocker platefor each sample that underwent agitation.

pH Measurements.

The pH each sample was measured using a micro-pH probe. Before the startof analysis the pH probe was calibrated with three pH standards orderedfrom fisher. The pH values of the stability samples were measured bytransferring 604 of each stability sample to 100 μL PCR tube. Themicro-pH probe was then submerged into the sample and after the valuestabilized it was recorded.

UV Absorbance Spectroscopy.

UV spectroscopy was used to measure the protein concentration in thesamples. The mole extinction coefficient at 280 nm for bulk substancewas 1.6355 mg/mL, which was determined experiential. The proteinconcentrations of the all formulations for LB-140 were measured using acell path length of 0.0096 cm. Below is the analysis parameters used forLB-140.

Scan Range: 400 to 200 nm

Average Time (min): 0.1

Date Interval (nm): 1

Scan Rate (nm/min): 600

Cycle Count: 5

Size Exclusion Chromatography (SEC) Method.

The SEC method used to analyze the LB-140 stability samples wasdeveloped at Legacy BioDesign. Below is a brief summary of the SECmethod parameter used for the analysis of the LB-140 samples.

Method Parameters

-   -   Column Information: ACQUITY UPLC BEH200 SEC, 1.7 urn Column,        4.6×150 mm    -   Analysis Buffer: 50 mM Phosphate, 250 mM NaCl, pH 6.8    -   Flow rate: 0.3 mL/min    -   Column temperature: 30° C.    -   Detection: 280 nm    -   Injection volume: 2 μL    -   Sample temperature: Approx. 5° C.

RP HPLC Method.

The RP HPLC method was found to be stability indicating and was used toanalyze LB-140 stability samples. Below is a summary of the RP methodparameter used for the analysis of the LB-140.

Method Parameters

-   -   Column Information: Poroshell 300SB-C8, 2.1×75 mm, 5 um    -   Mobile Phase A: 98% (v/v) H2O/2% (v/v) IPA/0.1% (v/v) TFA    -   Mobile Phase B: 10% (v/v) H2O/70% (v/v) IPA/20% (v/v) ACN/0.1%        (v/v) TFA    -   Flow rate: 0.25 mL/min    -   Column temperature: 80° C.    -   Detection: 225 nm    -   Injection volume: 1 μL    -   Sample temperature: Approx. 5° C.    -   Run time: 15 minutes        -   Gradient:

Time % A % B 0 100 0 10 50 50 10.1 100 0 15 100 0

CE-IEF Analysis.

Capillary isoelectric focusing (cIEF) was conducted as described in thePA 800 plus Application Guide published by Beckman Coulter. A moredetailed description can be found in a research article published byMack et al¹. All analyses were conducted using a Beckman Coulter P/ACEMDQ system (Beckman Coulter, Inc.; Brea, Calif.) operated at ambienttemperature with a 30 cm total length (20 cm effective) neutralcapillary. The neutral capillary was prepared by immobilizingpoly(acrylamide) to the capillary wall using a method described by Gaoet al.² cIEF samples were prepared by mixing the protein of interest at0.25 mg/mL with a mixture of 3M urea-cIEF gel containing ampholyte,cathodic stabilizer, anodic stabilizer, and pI markers. Sample waspressure injected at 9.5 psi into the capillary for 4.1 min, after whichtime it was focused by applying a voltage of 25 kV for 15 min betweenanalyte and catholyte. This step was followed by chemical mobilizationat 30 kV for 30 min between analyte and chemical mobilizer. The pImarkers and the protein of interest were detected with absorbance at 280nm during the mobilization step. The pI of the protein was calculatedfrom the resultant regression equation of pI vs. first peak momentobtained from the pI standards.

CE-SDS Analysis.

Analysis by CE-SDS was conducted under reducing conditions utilizing amethod adapted from the SOP published by Beckman-Coulter for determiningIgG purity/heterogeneity. Briefly, the antibody was diluted with DDIwater to 6 mg/mL, denatured by adding sample buffer (0.1 M Tris/1.0%SDS, pH 8.0), and reduced via addition of 2-mercaptoethanol; the finalantibody concentration was 1.2 mg/mL. Denaturing and reduction wasfacilitated by heating the sample at 70° C. for 10 min. The sample wascooled for 10 min at room temperature prior to analysis. A centrifugestep (300 g, 5 min) was employed prior to heating the sample anddirectly after the cooling it. CE analysis was conducted using a BeckmanCoulter P/ACE MDQ system operated at ambient temperature with a 30 cmtotal length (20 cm effective, 50 μm i.d.) capillary. Prior to sampleintroduction, the capillary was sequentially rinsed with 0.1 M NaOH,0.1M HCL, DDI water, and SDS-gel buffer solution. Sample was injectedelectrokinetically at 5 kV for 30 s followed by separation at 30 kV for30 min. For both injection and separation, the instrument was operatedin reverse polarity mode. Antibody fragments were detected usingabsorbance at 214 nm (4 Hz acquisition) and time-normalized areasreported for measured peaks.

Block a Formulation Studies

The Block A studies examined different buffer systems and used acommercially available adalimumab material which was reprocessed forthese studies. We note that U.S. Pat. No. 8,216,583 references stabilityof an adalimumab formulation in relation to use of a citrate/phosphatebuffer system at pH 5.2, and in fact the patent required the use of sucha buffer combination. The work we have done, reflected here, indicatesthat citrate/phosphate is in fact a rather poor buffer choice incomparison to others such as histidine and succinate. In the Block Astudies below, pH was kept constant at 5.2. The concentrations ofmannitol and polysorbate 80 were also held constant. Samples were keptat 40° C. for two weeks. The study design is summarized in the Tablebelow.

TABLE A BLOCK A STUDY DESIGN Form Citrate Phosphate Succinate HistidineTartrate Maleate Mannitol PS No. API (mM) (mM) (mM) (mM) (mM) (mM) (mM)80% 1 Humira ® 8 18 0 0 0 0 12 0.1 2 Humira ® 10 0 0 0 0 0 12 0.1 3Humira ® 0 10 0 0 0 0 12 0.1 4 Humira ® 0 0 10 0 0 0 12 0.1 5 Humira ® 00 0 10 0 0 12 0.1 6 Humira ® 0 0 0 0 10 0 12 0.1 7 Humira ® 0 0 0 0 0 1012 0.1

Analysis by SEC showed that the formulation with citrate alone performedmore poorly than the buffer combination (Table A), indicating that thephosphate was the primary stabilizer in that combination. This wassurprising and unexpected, as this pH is outside of the nominalbuffering capacity range of phosphate, but well within the bufferingrange for citrate. Furthermore, succinate, histidine, and tartrate didas well or better than the citrate/phosphate combination, indicatingthat other buffer systems would provide equal or superior stability foradalimumab. Accordingly, the present invention in one of its embodimentsis directed to adalimumab formulations exhibiting long term stability,wherein a buffer combination of citrate and phosphate is avoided infavor of at least one buffer selected from the group consisting ofhistidine, phosphate, succinate and tartrate. Acetate is also a suitablereplacement for the citrate phosphate buffer combination.

The purity of these stored samples was checked using RP HPLC (FIG. 2).As with SEC, the citrate formulation exhibited the poorest stability,while all of the other buffers did as well or better than the buffercombination found in commercially available adalimumab (Humira®). Theseresults demonstrate our discovery that changing the buffer (i.e.avoiding the citrate/phosphate buffer combination of the commercialadalimumab) could improve the stability profile of adalimumab.

Block B Formulation Studies

A second study (“BLOCK B”) was conducted examining just changes in thebuffer species, where the pH (5.2) was not changed, as outlined in thetable below labeled “BLOCK B Study Design. In this case, thecommercially available formulation for Humira® was used as a control,while all of the other formulations employed a proprietary adalimumabbiosimilar protein. Table B-1, below summarizes the percent monomer forthe Block B formulations (as well the percentage amount of an impuritydetermined to be a fragment of the adalimumab protein.

TABLE B BLOCK B STUDY DESIGN Form Citrate Phosphate Succinate HistidineTartrate Maleate Mannitol PS 80 No. API (mM) (mM) (mM) (mM) (mM) (mM)(mM) (%) 1 Humira ® 8 18 0 0 0 0 12 0.1 2 Adalimumab 10 0 0 0 0 0 12 0.1biosimilar 3 Adalimumab 0 10 0 0 0 0 12 0.1 biosimilar 4 Adalimumab 0 010 0 0 0 12 0.1 biosimilar 5 Adalimumab 0 0 0 10 0 0 12 0.1 biosimilar 6Adalimumab 0 0 0 0 10 0 12 0.1 biosimilar 7 Adalimumab 0 0 0 0 0 10 120.1 biosimilar

TABLE B-1 Percent monomer for Block B formulations at t0 and after twoweeks at 40 C. (t2) Form Monomer Monomer Fragment Fragment No. APIBuffer (t0) (t2) (t0) (t2) 1 Humira ® Citrate/ 99.34 0.26 phosphate 2Adalimumab citrate 98.71 97.92 0.62 0.40 biosimilar 3 Adalimumabphosphate 99.21 98.07 0.05 0.30 biosimilar 4 Adalimumab succinate 99.1998.04 0.04 0.31 biosimilar 5 Adalimumab histidine 99.19 98.41 0.07 0.23biosimilar 6 Adalimumab tartrate 99.13 98.10 0.04 0.29 biosimilar 7Adalimumab maleate 98.91 97.90 0.36 0.76 biosimilar

As can be seen from Table B-1 above, upon storage for two weeks at 40 C,the monomer content decreases by more than 1% for all of the samples inBlock B, except for the one containing histidine (His) buffer (TableB-1). From this study we discovered the likelihood that His would be asuperior buffer system for adalimumab. (We note that the fragment levelmeasured by SEC reported for Formulation 2 appears to be incorrect asall of the other initial fragment s levels are less than 0.1%.)

Block C Formulation Studies

A large-scale formulation screening study was carried out in the studiesconducted in Block C (See Table C, below). Samples were stored for oneweek at 40 C (hereinafter referenced as “t1”) or two weeks at 25 C(hereinafter referenced as “t2”). These conditions were used throughoutthe remainder of our studies, so this terminology will be employedthroughout the present detailed discussion. Block C was designed toexpand on the buffer assessment conducted in Block B. In addition, itexamined glycine (Gly) and/or arginine (Arg) as possible stabilizers inplace of mannitol and/or NaCl (Table C). Note that the buffer systemused in the Humira® product employs the 8 mM citrate/18 mM phosphatebuffer, which is the composition of Formulation 1 of Block C. In thiscase, a proprietary adalimumab biosimilar protein was used forformulation 1 of Block C, instead of adalimumab protein obtained fromcommercially available Humira®.

TABLE C BLOCK C STUDY DESIGN Form No. API citrate phosphate succinatehistidine glycine arginine mannitol NaCl 1 Adalimumab 8 18 0 0 0 0 65100 biosimilar 2 Adalimumab 18 8 0 0 0 0 65 100 biosimilar 3 Adalimumab20 0 0 0 0 0 65 100 biosimilar 4 Adalimumab 20 0 0 0 65 0 0 100biosimilar 5 Adalimumab 0 20 0 0 65 0 0 100 biosimilar 6 Adalimumab 20 00 0 0 65 0 100 biosimilar 7 Adalimumab 0 20 0 0 0 65 0 100 biosimilar 8Adalimumab 0 0 20 0 65 0 0 100 biosimilar 9 Adalimumab 0 0 20 0 0 65 0100 biosimilar 10 Adalimumab 0 0 0 20 65 0 0 100 biosimilar 11Adalimumab 0 0 0 20 0 65 0 100 biosimilar 12 Adalimumab 0 20 0 0 0 130 035 biosimilar 13 Adalimumab 0 0 20 0 0 130 0 35 biosimilar 14 Adalimumab0 0 0 20 0 130 0 35 biosimilar 15 Adalimumab 0 20 0 0 130 0 0 60biosimilar 16 Adalimumab 0 20 0 20 130 0 0 60 biosimilar

TABLE C-1 Measured pH for Block C formulations at t0 and t1 (one week,40° C.) Form pH pH No. citrate phosphate succinate histidine glycinearginine mannitol NaCl t0 t2 1 8 18 0 0 0 0 65 100 5.51 5.57 2 18 8 0 00 0 65 100 5.46 5.43 3 20 0 0 0 0 0 65 100 5.28 5.27 4 20 0 0 0 65 0 0100 5.27 5.24 5 0 20 0 0 65 0 0 100 5.43 5.44 6 20 0 0 0 0 65 0 100 5.295.29 7 0 20 0 0 0 65 0 100 5.28 5.32 8 0 0 20 0 65 0 0 100 5.22 5.17 9 00 20 0 0 65 0 100 5.19 5.16 10 0 0 0 20 65 0 0 100 5.28 5.30 11 0 0 0 200 65 0 100 5.26 5.29 12 0 20 0 0 0 130 0 35 5.24 5.24 13 0 0 20 0 0 1300 35 5.18 5.16 14 0 0 0 20 0 130 0 35 5.28 5.35 15 0 20 0 0 130 0 0 605.31 5.31 16 0 20 0 20 130 0 0 60 5.36 5.40

TABLE C-2 Monomer content by SEC for formulations in Block C at t0, t1(one week at 40° C.), and t2 (two weeks at 25° C.) Form No. citratephosphate succinate His Gly Arg mannitol NaCl t0 t1 t2 1 8 18 0 0 0 0 65100 98.75 97.90 98.06 2 18 8 0 0 0 0 65 100 99.26 98.22 98.80 3 20 0 0 00 0 65 100 99.28 98.32 98.78 4 20 0 0 0 65 0 0 100 99.36 98.45 99.03 5 020 0 0 65 0 0 100 99.25 98.20 98.77 6 20 0 0 0 0 65 0 100 99.42 98.6899.10 7 0 20 0 0 0 65 0 100 99.39 98.59 99.13 8 0 0 20 0 65 0 0 10099.41 98.51 99.04 9 0 0 20 0 0 65 0 100 99.36 98.52 98.96 10 0 0 0 20 650 0 100 99.41 98.66 99.15 11 0 0 0 20 0 65 0 100 99.37 98.70 99.15 12 020 0 0 0 130 0 35 99.41 98.66 99.14 13 0 0 20 0 0 130 0 35 99.42 98.7199.17 14 0 0 0 20 0 130 0 35 99.40 98.75 99.26 15 0 20 0 0 130 0 0 6099.32 98.53 99.05 16 0 20 0 20 130 0 0 60 99.40 98.66 99.19

TABLE C-3 Percent purity by RP HPLC for formulations in Block C at t0,t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. citratephosphate succinate His Gly Arg mannitol NaCl t0 t1 t2 1 8 18 0 0 0 0 65100 98.04 97.92 98.10 2 18 8 0 0 0 0 65 100 97.94 97.83 98.03 3 20 0 0 00 0 65 100 98.03 97.92 98.00 4 20 0 0 0 65 0 0 100 97.94 97.75 97.98 5 020 0 0 65 0 0 100 97.98 97.69 97.95 6 20 0 0 0 0 65 0 100 97.89 97.7297.92 7 0 20 0 0 0 65 0 100 97.80 97.70 97.91 8 0 0 20 0 65 0 0 10097.98 97.77 98.01 9 0 0 20 0 0 65 0 100 97.98 97.73 97.94 10 0 0 0 20 650 0 100 97.98 97.76 98.00 11 0 0 0 20 0 65 0 100 97.87 97.78 97.97 12 020 0 0 0 130 0 35 97.88 97.71 97.95 13 0 0 20 0 0 130 0 35 97.95 97.6297.93 14 0 0 0 20 0 130 0 35 97.98 97.72 98.04 15 0 20 0 0 130 0 0 6097.91 97.72 97.96 16 0 20 0 20 130 0 0 60 98.00 97.79 97.78

TABLE C-4 Percentage of main bands seen in the cIEF profile offormulations in Block C at t0, t1 (one week at 40° C.), and t2 (twoweeks at 25° C.) Form No. pH t0 t1 t2 1 8.59 1.94 1.97 1.82 8.43 11.7611.30 12.49 8.27 58.29 49.88 51.54 8.20 7.18 7.59 8.05 21.49 22.38 19.797.86 6.53 5.35 4.66 2 8.60 1.96 1.84 8.44 12.08 10.89 8.29 51.70 47.638.22 9.74 12.32 8.09 16.29 18.25 7.91 3.50 3.64 3 8.60 1.83 1.82 1.128.43 11.58 9.67 10.40 8.27 45.80 32.99 44.04 8.20 12.44 22.27 18.68 8.0117.57 16.21 14.40 7.86 4.39 3.61 4 8.57 2.31 2.04 2.13 8.41 12.94 11.5112.62 8.25 33.37 59.98 61.97 8.20 23.03 8.02 15.21 18.33 16.07 7.88 3.455.32 3.70 5 8.58 2.40 2.00 2.30 8.41 13.01 11.02 12.34 8.25 42.09 46.3237.30 8.21 15.58 10.65 15.80 8.03 18.48 20.58 16.80 7.86 3.74 6.13 4.836 8.57 2.83 8.38 13.17 13.23 8.23 32.66 31.18 8.18 17.52 18.54 8.0217.48 13.82 7.91 5.30 5.83 7 8.58 2.08 2.41 2.64 8.44 13.42 12.64 12.638.27 56.79 52.48 54.76 8.16 5.36 6.16 6.38 8.04 16.91 20.09 18.45 7.945.44 4.12 5.15 8 8.57 1.76 2.37 1.55 8.44 14.41 12.13 11.61 8.29 60.0148.87 52.94 8.19 7.07 10.66 8.10 16.22 16.55 17.10 7.95 7.61 5.02 4.55 98.58 2.19 2.06 0.99 8.41 11.69 10.64 12.73 8.26 50.07 44.21 60.33 8.1910.66 10.39 8.01 15.62 21.51 17.79 7.87 4.67 5.37 8.16 10 8.57 1.78 2.641.62 8.41 10.55 10.95 8.11 8.25 43.82 42.93 36.11 8.21 15.96 15.24 17.668.02 14.63 14.58 14.22 7.88 3.82 4.21 3.95 11 8.58 1.59 1.81 1.89 8.4112.98 11.58 12.86 8.23 62.74 29.63 12.00 8.19 22.86 34.77 8.02 17.1519.52 17.06 7.87 5.54 5.56 4.77 12 8.61 0.35 1.57 1.47 8.35 13.24 13.418.83 8.19 43.18 60.12 26.52 8.15 15.43 20.46 25.60 7.98 16.74 17.38 7.884.96 4.44 4.99 13 8.58 1.71 1.67 8.41 11.63 10.01 8.26 49.19 42.65 8.2014.25 16.64 8.03 17.35 18.12 7.86 4.28 4.18 14 8.56 1.64 1.79 1.73 8.3913.17 10.45 10.96 8.25 58.68 46.06 45.60 8.21 11.03 13.34 8.07 14.1020.24 14.50 7.92 2.10 5.13 4.28 15 8.57 1.74 1.22 1.60 8.41 10.49 15.2110.78 8.25 46.06 55.05 44.98 8.20 14.46 13.79 8.02 13.90 20.31 10.797.89 4.23 4.90 3.43 16 8.56 1.96 1.08 8.40 9.25 12.23 12.58 8.24 38.0831.03 58.61 8.20 19.02 22.08 21.50 8.03 12.00 13.24 7.31 7.89 4.73 4.82

TABLE C-5 Percentage of bands for light chain (LC), heavy chain (HC),non-glycosylated HC, and other species for formulations in Block C att0, t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. TimeLC HC ngHC Other 1 t0 35.87 63.20 0.51 0.42 t1 29.71 63.08 0.37 6.84 t231.01 67.83 0.54 0.61 2 t0 29.50 69.57 0.56 0.37 t1 30.51 67.28 0.561.65 t2 32.32 65.51 0.56 1.61 3 t0 32.53 66.45 0.54 0.47 t1 33.04 65.340.55 1.07 t2 31.94 66.60 0.57 0.90 4 t0 33.40 64.90 0.46 1.24 t1 30.9667.16 0.52 1.36 t2 32.08 65.84 0.56 1.52 5 t0 34.17 63.89 0.49 1.45 t133.60 64.27 0.56 1.57 t2 32.15 66.20 0.48 1.17 6 t0 37.91 60.35 0.541.19 t1 34.80 62.88 0.73 1.59 t2 32.90 65.62 0.50 0.99 7 t0 32.17 66.800.55 0.49 t1 29.83 68.33 0.59 1.25 t2 33.32 65.97 0.55 0.15 8 t0 33.8365.51 0.49 0.17 t1 30.37 68.48 0.58 0.57 t2 32.86 66.40 0.55 0.19 9 t030.69 69.31 0.00 0.00 t1 34.30 64.24 0.52 0.94 t2 29.08 69.87 0.62 0.4310 t0 38.68 59.95 0.57 0.80 t1 36.52 58.65 0.00 4.83 t2 43.68 54.39 1.920.00 11 t0 35.25 59.00 1.75 4.00 t1 30.71 67.58 0.66 1.05 t2 30.18 67.140.47 2.21 13 t0 44.58 55.42 0.00 0.00 t1 37.73 60.75 0.25 1.28 t2 38.0561.44 0.52 0.00 14 t0 32.50 66.66 0.60 0.24 t1 30.91 67.77 0.61 0.70 t229.14 70.32 0.23 0.31 15 t0 30.07 68.95 0.63 0.35 t1 30.14 68.49 0.620.75 t2 31.57 67.55 0.62 0.26 16 t0 30.54 68.61 0.63 0.22 t1 29.81 68.810.63 0.75 t2 29.46 69.14 0.59 0.81

Discussion of Block C Results

Referring to Table C-1 above, the pH was measured and found to berelatively stable for all of the formulations. However, the initial pHvalues were slightly higher for the citrate/phosphate formulations. Theleast stable formulation by SEC analysis appears to be Formulation 1,the one using the Humira® buffer system. By comparison we discoveredthat formulations using His as the buffer and/or formulations containingGly or Arg exhibited the greatest stability (See Table C-2). Similartrends are seen when the purity by RP HPLC is considered (See TableC-3). It appears that SEC may be a better stability-indicating methodthan RP HPLC, although, when taken as a whole, the RP HPLC method doesappear to be stability-indicating. Based on the Block C data summarizedabove, we have discovered that Histidine is suitable as a preferredbuffer in terms of formulation stability, and that glycine or arginine,or combinations thereof, are also stability enhancing components forinclusion in an adalimumab formulation.

The stored samples were further analyzed by cIEF at t1 and t2 (Table C-4above). A proprietary adalimumab material exhibit four to five peakswith integrated intensities above 1% or so. In general, there are somesmall decreases in the intensity of the main peak upon storage. Theselosses are usually greater at t1 than at t2. Still, no significant newpeaks are observed, suggesting that there is minimal chemicaldegradation occurring that would lead to changes in the overall chargeon the protein. The variance in the data indicates that this method,while useful for characterization, does not appear to bestability-indicating.

The final analytical method used to evaluate the stability of adalimumabformulation is CE-SDS, which is essentially the CE version of SDS-PAGEslab gels. This method indicates that the relative areas of the LC peakdo decrease when stored at elevated temperatures (Table C-5), while theamount of new peaks (cumulatively called ‘Other’) increases. Altogether,these changes are usually less than 2% for any of the formulations.There are some samples where the percentage of ‘Other’ is in the 4-6%range, but these are likely artifacts.

Block D Formulation Studies

Another set of formulations were evaluated as “Block D.” Sixteenformulations were designed to evaluate other stabilizers as alternativesto mannitol, such as sorbitol and trehalose (See Table D). Block D alsoexamined using mannitol or NaCl as the sole tonicity agent, instead ofusing a mixture of the two excipients. The pH stability of theformulations was quite good, although the actual initial pH values wereslightly lower than the target values for some formulations (Table D-1).

TABLE D BLOCK D STUDY DESIGN Form PS No. API citrate phosphate sorbitoltrehalose mannitol NaCl 80 1 Adalimumab 8 18 0 0 65 100 0.1 biosimilar 2Adalimumab 8 18 0 0 65 100 0 biosimilar 3 Adalimumab 20 0 0 0 65 100 0.1biosimilar 4 Adalimumab 20 0 0 0 65 100 0 biosimilar 5 Adalimumab 0 20 00 65 100 0.1 biosimilar 6 Adalimumab 0 20 0 0 65 100 0 biosimilar 7Adalimumab 8 18 65 0 0 100 0.1 biosimilar 8 Adalimumab 8 18 0 65 0 1000.1 biosimilar 9 Adalimumab 0 20 65 0 0 100 0.1 biosimilar 10 Adalimumab0 10 0 0 240 0 0.1 biosimilar 11 Adalimumab 0 10 240 0 0 0 0.1biosimilar 12 Adalimumab 0 10 0 240 0 0 0.1 biosimilar 13 Adalimumab 100 0 0 0 150 0.1 biosimilar 14 Adalimumab 10 0 0 0 0 150 0 biosimilar 15Adalimumab 0 10 0 0 0 150 0.1 biosimilar 16 Adalimumab 0 10 0 0 0 150 0biosimilar

TABLE D-1 Measured pH for Block D formulations at t0 and t1 (one week,40° C.) Form PS No. API citrate phosphate sorbitol trehalose mannitolNaCl 80 t0 t1 t2 1 Adalimumab 8 18 0 0 65 100 0.1 5.09 5.17 5.12biosimilar 2 Adalimumab 8 18 0 0 65 100 0 5.12 5.16 5.16 biosimilar 3Adalimumab 20 0 0 0 65 100 0.1 5.11 5.16 5.14 biosimilar 4 Adalimumab 200 0 0 65 100 0 5.13 5.17 5.18 biosimilar 5 Adalimumab 0 20 0 0 65 1000.1 5.19 5.25 5.24 biosimilar 6 Adalimumab 0 20 0 0 65 100 0 5.16 5.245.17 biosimilar 7 Adalimumab 8 18 65 0 0 100 0.1 5.14 5.17 5.18biosimilar 8 Adalimumab 8 18 0 65 0 100 0.1 5.15 5.21 5.16 biosimilar 9Adalimumab 0 20 65 0 0 100 0.1 5.19 5.29 5.28 biosimilar 10 Adalimumab 010 0 0 240 0 0.1 5.23 5.28 5.27 biosimilar 11 Adalimumab 0 10 240 0 0 00.1 5.45 5.35 5.33 biosimilar 12 Adalimumab 0 10 0 240 0 0 0.1 5.44 5.325.31 biosimilar 13 Adalimumab 10 0 0 0 0 150 0.1 5.30 5.25 5.23biosimilar 14 Adalimumab 10 0 0 0 0 150 0 5.39 5.20 5.18 biosimilar 15Adalimumab 0 10 0 0 0 150 0.1 5.35 5.30 5.22 biosimilar 16 Adalimumab 010 0 0 0 150 0 5.41 5.33 5.28 biosimilar

TABLE D-2 Monomer content by SEC for formulations in Block D at t0, t1(one week at 40° C.), and t2 (two weeks at 25° C.) Form PS No. APIcitrate phosphate sorbitol trehalose mannitol NaCl 80 t0 t1 t2 1Adalimumab 8 18 0 0 65 100 0.1 99.28 98.21 98.96 biosimilar 2 Adalimumab8 18 0 0 65 100 0 99.25 98.11 98.85 biosimilar 3 Adalimumab 20 0 0 0 65100 0.1 99.25 98.16 98.86 biosimilar 4 Adalimumab 20 0 0 0 65 100 099.27 98.26 98.92 biosimilar 5 Adalimumab 0 20 0 0 65 100 0.1 99.2498.16 98.84 biosimilar 6 Adalimumab 0 20 0 0 65 100 0 99.21 98.23 98.82biosimilar 7 Adalimumab 8 18 65 0 0 100 0.1 99.30 98.19 98.94 biosimilar8 Adalimumab 8 18 0 65 0 100 0.1 99.28 98.14 98.85 biosimilar 9Adalimumab 0 20 65 0 0 100 0.1 99.29 98.23 98.90 biosimilar 10Adalimumab 0 10 0 0 240 0 0.1 97.93 98.54 biosimilar 11 Adalimumab 0 10240 0 0 0 0.1 99.32 98.65 99.00 biosimilar 12 Adalimumab 0 10 0 240 0 00.1 99.32 98.53 98.96 biosimilar 13 Adalimumab 10 0 0 0 0 150 0.1 99.2998.12 98.84 biosimilar 14 Adalimumab 10 0 0 0 0 150 0 99.28 98.28 98.90biosimilar 15 Adalimumab 0 10 0 0 0 150 0.1 99.26 97.99 98.83 biosimilar16 Adalimumab 0 10 0 0 0 150 0 99.20 97.76 98.62 biosimilar

TABLE D-3 Percent purity by RP HPLC for formulations in Block D at t0,t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form PS No. APIcitrate phosphate sorbitol trehalose mannitol NaCl 80 t0 t1 t2 1Adalimumab 8 18 0 0 65 100 0.1 98.17 97.75 98.02 biosimilar 2 Adalimumab8 18 0 0 65 100 0 98.09 97.84 98.08 biosimilar 3 Adalimumab 20 0 0 0 65100 0.1 98.03 97.81 98.19 biosimilar 4 Adalimumab 20 0 0 0 65 100 098.17 97.85 98.06 biosimilar 5 Adalimumab 0 20 0 0 65 100 0.1 98.1197.88 98.18 biosimilar 6 Adalimumab 0 20 0 0 65 100 0 98.21 97.77 98.10biosimilar 7 Adalimumab 8 18 65 0 0 100 0.1 98.11 97.80 98.14 biosimilar8 Adalimumab 8 18 0 65 0 100 0.1 98.06 97.73 98.03 biosimilar 9Adalimumab 0 20 65 0 0 100 0.1 98.09 97.80 98.07 biosimilar 10Adalimumab 0 10 0 0 240 0 0.1 98.13 97.82 98.08 biosimilar 11 Adalimumab0 10 240 0 0 0 0.1 98.10 97.90 98.06 biosimilar 12 Adalimumab 0 10 0 2400 0 0.1 98.13 97.95 98.14 biosimilar 13 Adalimumab 10 0 0 0 0 150 0.198.07 97.79 98.02 biosimilar 14 Adalimumab 10 0 0 0 0 150 0 98.13 97.7898.14 biosimilar 15 Adalimumab 0 10 0 0 0 150 0.1 98.17 97.80 98.10biosimilar 16 Adalimumab 0 10 0 0 0 150 0 98.14 97.79 98.06 biosimilar

TABLE D-4 Percentage of main bands seen in the cIEF profile offormulations in Block D at t0, t1 (one week at 40° C.), and t2 (twoweeks at 25° C.) Form No. pH t0 t1 t2 1 8.56 2.26 1.81 8.41 13.84 12.8811.73 8.25 62.27 59.80 56.15 8.14 6.48 8.04 15.71 22.93 13.73 7.99 5.924.39 4.13 2 8.55 2.08 1.58 8.40 12.89 12.58 8.24 60.15 53.24 8.14 5.986.69 8.03 11.92 9.72 7.98 3.65 5.67 3 8.57 1.58 2.10 1.89 8.41 11.8711.83 11.99 8.26 54.93 54.45 54.51 8.16 9.10 6.31 8.24 8.05 9.21 11.1610.22 7.91 7.60 4.16 5.26 4 8.57 3.57 1.82 1.05 8.40 11.12 10.66 10.838.24 49.37 47.85 42.34 8.14 3.01 1.83 3.68 8.03 10.11 10.06 17.12 7.902.78 4.72 3.84 5 8.55 2.30 2.18 2.13 8.40 7.63 8.86 8.63 8.25 33.9014.41 16.64 8.20 23.41 33.90 33.75 8.03 10.14 20.39 19.42 7.99 6.76 5.424.63 6 8.59 1.87 1.39 8.42 11.25 11.18 11.89 8.27 50.07 61.72 64.17 8.2012.43 22.08 19.18 8.03 10.20 7.91 2.70 5.01 3.38 7 8.55 8.40 8.25 8.208.03 7.99 8 8.59 1.46 2.64 1.16 8.39 13.52 13.62 7.37 8.22 60.79 50.8355.40 8.08 5.21 11.28 9.78 8.02 15.24 8.55 11.94 7.91 3.79 3.02 5.18 98.53 2.64 3.25 1.94 8.38 13.83 12.72 11.67 8.25 64.97 51.32 54.14 8.178.33 11.21 8.61 8.06 11.75 9.98 9.03 8.01 5.79 4.80 7.31 10 8.54 1.783.26 8.38 13.04 11.19 8.21 60.53 44.83 8.15 19.60 10.95 7.99 9.41 7.905.05 4.27 11 8.52 1.95 2.11 1.89 8.36 11.24 12.43 12.43 8.21 48.64 54.1059.90 8.13 11.69 6.31 8.00 10.30 21.14 11.14 8.01 5.27 5.64 8.32 12 8.511.85 8.29 11.31 11.38 8.18 63.11 45.14 8.14 2.54 8.05 16.16 22.03 7.945.03 6.88 13 8.62 3.51 3.05 8.44 12.44 12.30 8.29 65.10 51.44 8.21 12.188.06 15.37 17.25 7.91 3.58 3.77 14 8.61 2.74 1.73 8.43 10.60 12.19 8.2746.23 41.11 8.21 13.97 10.49 8.05 18.56 17.52 7.91 5.15 15 8.62 8.3512.40 10.91 8.34 8.21 31.87 30.32 36.39 8.20 41.14 25.57 30.62 8.0212.42 13.72 18.26 7.89 2.18 5.44 3.86 16 8.61 8.48 12.96 12.86 13.198.34 34.40 31.45 39.25 8.31 27.74 20.29 18.81 8.05 22.76 19.35 7.89 8.177.69 4.83

TABLE D-5 Percentage of bands for light chain (LC), heavy chain (HC),non-glycosylated HC, and other species for formulations in Block D att0, t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. TimeLC HC ngHC Other 1 t0 t1 34.11 62.58 0.58 2.73 t2 33.19 64.28 0.60 1.922 t0 30.25 66.81 0.64 2.31 t1 30.61 65.79 0.54 3.07 t2 29.22 67.04 0.643.10 3 t0 27.48 68.51 0.59 3.42 t1 30.84 67.27 0.54 1.35 t2 30.30 68.130.58 0.99 4 t0 30.88 68.33 0.60 0.19 t1 29.76 68.32 0.57 1.34 t2 31.4966.95 0.55 1.01 5 t0 33.77 64.50 0.56 1.17 t1 31.59 66.54 0.52 1.34 t229.19 69.16 0.59 1.06 6 t0 30.90 68.08 0.56 0.47 t1 29.32 69.88 0.540.26 t2 31.08 67.58 0.54 0.79 7 t0 30.41 68.60 0.56 0.43 t1 30.87 66.950.55 1.63 t2 30.14 68.28 0.55 1.03 8 t0 31.68 67.41 0.60 0.31 t1 t2 9 t029.62 68.12 0.51 1.75 t1 t2 29.46 68.10 0.61 1.83 10 t0 29.80 67.99 0.581.64 t1 30.04 65.53 0.45 3.98 t2 30.41 66.27 0.53 2.80 11 t0 29.85 67.630.61 1.91 t1 29.02 68.18 0.60 2.20 t2 30.44 67.14 0.58 1.84 12 t0 29.3868.11 0.55 1.96 t1 30.16 65.55 0.49 3.80 t2 28.20 69.19 0.59 2.02 13 t031.38 66.28 0.55 1.79 t1 33.67 64.10 0.56 1.67 t2 29.72 67.99 0.58 1.7114 t0 37.34 60.53 0.52 1.62 t1 33.03 63.46 0.53 2.97 t2 34.39 63.62 0.541.45 15 t0 30.20 68.42 0.59 0.79 t1 28.67 69.42 0.58 1.33 t2 29.96 68.240.56 1.24 16 t0 31.62 66.95 0.58 0.85 t1 30.48 66.36 0.55 2.61 t2 27.9470.17 0.60 1.29

Results of Block D

The pH stability was quite good for these formulations (Table D-1). Onceagain, the commercial adalimumab (Humira®) formulation was used as acontrol (but using a proprietary adalimumab biosimilar protein as theAPI). The commercial formulation again showed poorer stability by SECthan those using single buffers like phosphate and His (See Table D-2).Of the two buffers used in Humira®, we have now discovered thatphosphate is the better stabilizer. This is surprising, as phosphate hasvirtually no buffer capacity at pH 5.2, while citrate buffers well atthis pH. This suggests that the differences in stability profile may bedue to direct interaction of the buffer with the protein, a phenomenonthat, in the case of the commercial Humira® formulation, we believe wasnot previously understood or appreciated. Accordingly, the comparativebenefit of selecting phosphate as a buffer in an adalimumab formulation,due to superior stability in the formulation versus the selection of acitrate/phosphate combination constitutes one of the important aspectsof our invention.

Both sorbitol and trehalose display better stability profiles thanmannitol when used as the sole tonicity agent in these formulations. Italso appears that removal of the polysorbate 80 (PS 80) decreasesstability somewhat. The best stability profile by SEC appears to be forFormulations 10 and 11, which contain high concentrations of sorbitol ortrehalose in place of mannitol/NaCl (Table D-2). These results indicateto us that removing NaCl from the formulation, or limiting itsconcentration below certain targeted levels (for example less than about100 mM), will be beneficial for stability. (We note that mannitol doesappear to be a stabilizing ingredient, but at levels preferably above150.)

The RP data indicates that either citrate or phosphate provides betterstability than the combination used in Humira® (Table D-3). Again, theavoidance of the citrate/phosphate combination represents an importantfeature of our invention. It could not have been known or predicted thatcitrate alone, or phosphate alone would provide better formulationstability than the commercial buffer system comprising a combination ofcitrate and phosphate.

The cIEF analyses were run for Block D samples (Table D-4 above). Asbefore, there is some decrease in the intensity of the main peak, but nonew peaks are observed. In some cases, there is some small increase inthe intensity of the more acidic peaks. The decreases in the main peakappear to be greater at t1 than at t2, suggesting that degradation at 5°C. would be almost imperceptible. Still, overall it looks like less than5% (and probably much less than 5%) is degrading as measured by cIEF(Table D-4). Likewise, little degradation is seen by CE-SDS (Table D-5).At most 2 to 4% degradation is seen, but the variability in the methodmakes it difficult to determine if these are real changes. There doesappear to be higher impurity levels (Other) for Formulations 1 and 2 and10 through 14.

Block E Formulation Studies

This block of formulations was designed to evaluate the stability offormulations at different pH levels. If a buffer is not specified,acetate buffer (10 mM) was employed (Table E). A secondary objective wasto evaluate Gly and Arg at higher concentrations and in combination asalternative stabilizers to mannitol and NaCl.

TABLE E BLOCK E STUDY DESIGN Form PS No. API pH citrate phosphatesorbitol Gly Arg mannitol NaCl 80 1 Adalimumab 5.2 8 18 0 0 0 65 100 0.1biosimilar 2 Adalimumab 3.5 8 18 0 0 0 65 100 0.1 biosimilar 3Adalimumab 5.2 0 0 0 0 0 65 100 0.1 biosimilar 4 Adalimumab 3.5 0 0 0 00 65 100 0.1 biosimilar 5 Adalimumab 3.5 0 0 65 0 0 0 100 0.1 biosimilar6 Adalimumab 3.5 0 0 0 0 130 0 0 0.1 biosimilar 7 Adalimumab 3.5 0 0 0 0130 0 0 0 biosimilar 8 Adalimumab 3.5 0 0 0 240 0 0 0 0 biosimilar 9Adalimumab 5.2 0 0 0 240 0 0 0 0 biosimilar 10 Adalimumab 3.5 0 0 0 100100 0 0 0 biosimilar 11 Adalimumab 5.2 0 0 0 100 100 0 0 0 biosimilar 12Adalimumab 3.5 0 0 0 150 50 0 0 0 biosimilar

TABLE E-1 Measured pH for Block E formulations at t0 and t1 (one week,40° C.) Form PS No. pH citrate phosphate sorbitol Gly Arg mannitol NaCl80 t0 t1 t2 1 5.2 8 18 0 0 0 65 100 0.1 5.15 5.11 5.21 2 3.5 8 18 0 0 065 100 0.1 3.36 3.49 3.50 3 5.2 0 0 0 0 0 65 100 0.1 5.13 5.24 5.24 43.5 0 0 0 0 0 65 100 0.1 3.31 3.43 3.45 5 3.5 0 0 65 0 0 0 100 0.1 3.303.48 3.42 6 3.5 0 0 0 0 130 0 0 0.1 3.24 3.52 3.42 7 3.5 0 0 0 0 130 0 00 3.27 3.59 3.48 8 3.5 0 0 0 240 0 0 0 0 3.27 3.33 3.39 9 5.2 0 0 0 2400 0 0 0 5.05 5.25 5.20 10 3.5 0 0 0 100 100 0 0 0 3.30 3.45 3.41 11 5.20 0 0 100 100 0 0 0 5.20 5.38 5.39 12 3.5 0 0 0 150 50 0 0 0 3.24 3.383.37

TABLE E-2 Monomer content by SEC for formulations in Block E at t0, t1(one week at 40° C.), and t2 (two weeks at 25° C.) Form PS No. pHcitrate phosphate sorbitol Gly Arg mannitol NaCl 80 t0 t1 t2 1 5.2 8 180 0 0 65 100 0.1 99.23 98.20 98.85 2 3.5 8 18 0 0 0 65 100 0.1 98.8244.15 86.37 3 5.2 0 0 0 0 0 65 100 0.1 99.30 98.37 99.02 4 3.5 0 0 0 0 065 100 0.1 95.85 33.51 76.21 5 3.5 0 0 65 0 0 0 100 0.1 97.37 26.2177.80 6 3.5 0 0 0 0 130 0 0 0.1 97.79 35.67 65.83 7 3.5 0 0 0 0 130 0 00 99.00 55.51 90.60 8 3.5 0 0 0 240 0 0 0 0 99.24 75.60 98.24 9 5.2 0 00 240 0 0 0 0 99.08 98.63 99.18 10 3.5 0 0 0 100 100 0 0 0 99.28 51.0391.66 11 5.2 0 0 0 100 100 0 0 0 99.32 98.54 99.09 12 3.5 0 0 0 150 50 00 0 99.29 45.86 93.06

TABLE E-3 Percent purity by RP HPLC for formulations in Block E at t0,t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form PS No. pHcitrate phosphate sorbitol Gly Arg mannitol NaCl 80 t0 t1 t2 1 5.2 8 180 0 0 65 100 0.1 98.58 96.88 96.91 2 3.5 8 18 0 0 0 65 100 0.1 98.5190.29 95.99 3 5.2 0 0 0 0 0 65 100 0.1 98.50 96.90 96.83 4 3.5 0 0 0 0 065 100 0.1 98.56 91.18 95.55 5 3.5 0 0 65 0 0 0 100 0.1 98.45 90.9695.71 6 3.5 0 0 0 0 130 0 0 0.1 98.71 93.28 95.38 7 3.5 0 0 0 0 130 0 00 98.40 90.65 96.54 8 3.5 0 0 0 240 0 0 0 0 98.03 93.94 96.82 9 5.2 0 00 240 0 0 0 0 98.23 97.19 97.12 10 3.5 0 0 0 100 100 0 0 0 98.13 91.1096.67 11 5.2 0 0 0 100 100 0 0 0 98.13 97.17 97.12 12 3.5 0 0 0 150 50 00 0 98.07 93.40 96.48

TABLE E-4 Percentage of bands for light chain (LC), heavy chain (HC),non-glycosylated HC, and other species for formulations in Block E att0, t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. TimeLC HC ngHC Other 1 t0 29.97 68.80 0.59 0.64 t1 28.49 70.07 0.60 0.81 t228.21 70.29 0.59 0.90 2 t0 28.50 68.67 0.52 2.31 t1 29.69 50.92 0.3019.09 t2 28.76 69.64 0.60 1.00 3 t0 24.30 74.01 0.60 1.09 t1 28.27 69.630.60 1.51 t2 28.17 69.89 0.54 1.40 4 t0 29.45 68.73 0.56 1.26 t1 29.5251.33 0.30 18.86 t2 27.92 65.73 0.52 5.83 5 t0 35.59 63.85 0.56 0.00 t132.47 48.72 0.30 18.52 t2 34.98 60.88 0.46 3.68 6 t0 34.33 63.39 0.511.77 t1 t2 35.32 61.31 0.45 2.92 7 t0 30.13 68.87 0.60 0.40 t1 28.1354.79 0.59 16.49 t2 34.39 63.32 0.53 1.76 8 t0 33.27 64.97 0.55 1.21 t133.20 52.62 0.33 13.85 t2 33.25 65.26 0.58 0.92 9 t0 32.28 66.34 0.570.81 t1 31.81 65.76 0.57 1.86 t2 31.23 66.81 0.57 1.39 10 t0 35.66 63.360.43 0.56 t1 24.96 58.61 0.33 16.10 t2 33.44 66.03 0.53 0.00 11 t0 29.7569.08 0.60 0.57 t1 27.67 70.83 0.61 0.89 t2 28.81 69.86 0.59 0.73 12 t030.23 49.07 0.26 20.44 t1 28.14 70.11 0.58 1.18 t2 29.75 69.08 0.60 0.57

TABLE E-5 Percentage of main bands seen in the cIEF profile offormulations in Block E at t0, t1 (one week at 40° C.), and t2 (twoweeks at 25° C. Form No. pH t0 t1 t2 1 8.56 8.37 12.52 12.65 8.23 51.7750.04 8.14 8.03 21.54 12.40 7.93 14.17 16.26 2 1.88 1.49 8.37 10.0717.66 14.15 8.21 37.52 32.26 33.88 8.13 19.03 9.96 8.01 16.57 28.70 7.934.12 7.45 4 8.54 1.04 2.67 8.38 10.50 9.32 8.21 68.34 31.91 8.13 28.528.02 16.55 10.05 7.88 3.57 8.67 5 6 7 8 9 8.60 1.40 2.60 3.26 8.43 10.0412.33 12.03 8.26 62.39 63.19 63.89 8.14 8.03 15.00 16.95 16.57 7.88 7.084.93 4.25

Results of Block E Studies

The pH stability was modest, with increases in pH occurring at t1 formany of the formulations, especially those buffered with acetate at lowpH (Table E-1 above). Two of the samples (Formulations 6 and 12) gelledat t1.

There were sizable losses in monomer content for the pH3.5 samples(Table E-3), whereas the pH 5.2 samples displayed stability comparableto what was seen in the preceding Blocks. It was also clear that thedegradation was much more pronounced at 40 C than at 25, despite beingstored for twice the length of time. In fact, Formulation 8 lost lessthan 1% monomer at t2 (Table E-2). The Gly and Arg formulations alldisplayed good stability, provided the pH was held 5.2. The data in thisblock of studies confirm our discovery that glycine or arginine, or amixture thereof are good stabilizers in an adalimumab formulation.

The RP HPLC data shows large decreases in purity, although not nearly asgreat as for monomer loss by SEC (Table E-3). This suggests thatchemical instability is less than physical instability. As with the SECresults, the loss of stability is more pronounced at t1 than at t2.

The CE-SDS results show large increases in new peaks, with the Othercategory increasing to 15-20% for low pH samples at t1 (Table E-4). Themost stable formulation by CE-SDS appears to be Formulation 11, whichcontains both Gly and Arg as the tonicity modifiers/stabilizers.

We encountered difficulties running the cIEF for many of the Block Esamples. However, given the clearly inferior stability at pH 3.5, it isunlikely that cIEF would provide any new information on those stabilityprofiles. For example, Formulation 4 (pH 3.5) shows a splitting of themain peak at t1.

Block F Formulation Studies

The Block F studies were intended to investigate the stability forHis-containing formulation using either mannitol, Gly or Arg as the soletonicity modifier (Table F below). It also served as an opportunity toevaluate additives such as EDTA and methionine (Met), which can beeffective at slowing oxidation. In addition, one high citrateconcentration and one high phosphate concentration formulation wereexamined.

TABLE F BLOCK F STUDY DESIGN Form PS No. API pH citrate phosphate HisGly Arg mannitol NaCl 80 EDTA Met 1 Adalimumab 5.2 8 18 0 0 0 65 100 0.10 0 biosimilar 2 Adalimumab 5.2 8 18 0 0 0 65 100 0.1 0.5 0 biosimilar 3Adalimumab 5.2 0 0 10 0 150 0 0 0 0.1 0 biosimilar 4 Adalimumab 5.2 0 010 0 150 0 0 0 0.5 0 biosimilar 5 Adalimumab 5.2 0 0 10 0 0 240 0 0 0 0biosimilar 6 Adalimumab 5.2 0 0 10 0 0 240 0 0 0 10 biosimilar 7Adalimumab 5.2 0 0 10 0 0 240 0 0 0 50 biosimilar 8 Adalimumab 5.2 30 00 240 0 0 0 0 0 0 biosimilar 9 Adalimumab 5.2 0 30 0 240 0 0 0 0 0 0biosimilar 10 Adalimumab 5.2 0 0 30 240 0 0 0 0 0 0 biosimilar 11Adalimumab 5.2 0 0 20 0 25 120 0 0.1 0 0 biosimilar 12 Adalimumab 5.2 00 20 0 25 120 0 0.1 0 0 biosimilar

TABLE F-1 Measured pH for Block F formulations at t0 and t1 (one week,40° C.) Form PS No. citrate phosphate His Gly Arg mannitol NaCl 80 EDTAMet t0 t1 t2 1 8 18 0 0 0 65 100 0.1 0 0 4.67 4.88 4.77 2 8 18 0 0 0 65100 0.1 0.5 0 5.05 5.15 5.20 3 0 0 10 0 150 0 0 0 0.1 0 5.11 5.22 5.27 40 0 10 0 150 0 0 0 0.5 0 4.95 5.06 5.15 5 0 0 10 0 0 240 0 0 0 0 5.125.25 5.29 6 0 0 10 0 0 240 0 0 0 10 4.45 4.74 4.67 7 0 0 10 0 0 240 0 00 50 5.03 5.24 5.24 8 30 0 0 240 0 0 0 0 0 0 5.09 5.18 5.22 9 0 30 0 2400 0 0 0 0 0 5.13 5.25 5.32 10 0 0 30 240 0 0 0 0 0 0 5.08 5.24 5.24 11 00 20 0 25 120 0 0.1 0 0 5.01 5.17 5.18 12 0 0 20 0 25 120 0 0.1 0 0 5.065.20 5.19

TABLE F-2 Monomer content by SEC for formulations in Block F at t0, t1(one week at 40° C.), and t2 (two weeks at 25° C.) Form PS No. citratephosphate His Gly Arg mannitol NaCl 80 EDTA Met t0 t1 t2 1 8 18 0 0 0 65100 0.1 0 0 97.69 94.75 96.06 2 8 18 0 0 0 65 100 0.1 0.5 0 99.25 98.1498.92 3 0 0 10 0 150 0 0 0 0.1 0 99.30 98.54 99.16 4 0 0 10 0 150 0 0 00.5 0 99.28 98.31 99.14 5 0 0 10 0 0 240 0 0 0 0 99.17 98.64 99.14 6 0 010 0 0 240 0 0 0 10 99.07 98.50 99.07 7 0 0 10 0 0 240 0 0 0 50 99.2998.92 99.24 8 30 0 0 240 0 0 0 0 0 0 99.28 98.40 99.04 9 0 30 0 240 0 00 0 0 0 99.30 98.50 99.08 10 0 0 30 240 0 0 0 0 0 0 99.31 98.60 99.23 110 0 20 0 25 120 0 0.1 0 0 99.27 98.64 99.16 12 0 0 20 0 25 120 0 0.1 0 099.29 98.51 99.17

TABLE F-3 Percent purity by RP HPLC for formulations in Block F at t0,t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form PS No.citrate phosphate His Gly Arg mannitol NaCl 80 EDTA Met t0 t1 t2 1 8 180 0 0 65 100 0.1 0 0 97.47 96.89 97.98 2 8 18 0 0 0 65 100 0.1 0.5 097.33 97.02 97.99 3 0 0 10 0 150 0 0 0 0.1 0 97.64 97.14 98.04 4 0 0 100 150 0 0 0 0.5 0 97.59 97.00 97.97 5 0 0 10 0 0 240 0 0 0 0 97.11 97.3098.03 6 0 0 10 0 0 240 0 0 0 10 97.61 97.27 98.03 7 0 0 10 0 0 240 0 0 050 97.55 97.37 98.08 8 30 0 0 240 0 0 0 0 0 0 97.48 97.51 98.05 9 0 30 0240 0 0 0 0 0 0 97.64 97.58 98.03 10 0 0 30 240 0 0 0 0 0 0 97.68 97.4198.06 11 0 0 20 0 25 120 0 0.1 0 0 97.67 97.18 98.03 12 0 0 20 0 25 1200 0.1 0 0 97.68 97.33 98.02

TABLE F-4 Percentage of bands for light chain (LC), heavy chain (HC),non- glycosylated HC, and other species for formulations in Block F att0, t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. TimeLC HC ngHC Other 1 t0 27.36 71.86 0.60 0.17 t1 t2 25.34 73.18 0.63 0.862 t0 27.80 71.07 0.60 0.53 t1 28.29 71.08 0.63 0.00 t2 27.53 70.97 0.640.86 3 t0 27.78 70.62 0.65 0.95 t1 28.26 70.85 0.66 0.23 t2 28.26 70.500.63 0.61 4 t0 28.20 70.24 0.60 0.96 t1 29.17 69.30 0.74 0.80 t2 29.1770.27 0.56 0.00 5 t0 27.50 70.74 0.59 1.17 t1 29.56 65.79 0.41 4.24 t228.24 69.90 0.58 1.28 6 t0 29.10 68.84 0.54 1.52 t1 28.58 69.18 0.541.70 t2 27.47 70.39 0.54 1.60 7 t0 27.87 70.28 0.55 1.30 t1 t2 8 t034.72 64.87 0.41 0.00 t1 34.94 64.53 0.53 0.00 t2 33.21 65.76 0.50 0.529 t0 31.96 68.04 0 0 t1 48.51 51.49 0 0 t2 33.15 65.82 0.57 0.46 10 t027.81 71.27 0.51 0.40 t1 29.59 68.46 0.53 1.43 t2 31.25 67.89 0.50 0.3611 t0 27.33 70.80 0.61 1.26 t1 26.54 71.00 0.64 1.82 t2 29.46 69.85 0.690.00 12 t0 24.18 71.21 0 4.61 t1 t2 28.95 68.98 0.59 1.46

TABLE F-5 Percentage of main bands seen in the cIEF profile offormulations in Block F at t0, t1 (one week at 40° C.), and t2 (twoweeks at 25° C. Form No. pH t0 t1 t2 1 8.65 2.11 8.31 13.07 11.43 34.178.24 64.66 67.99 28.67 8.14 2.29 8.08 16.13 17.73 15.98 7.95 6.14 4.80 28.60 1.56 1.93 1.12 8.48 12.95 11.26 11.12 8.24 58.99 55.85 60.37 8.1320.98 22.43 18.77 7.93 3.56 6.41 6.09 8.60 1.56 1.93 1.12 3 8.56 1.561.69 8.34 10.88 12.85 10.83 8.18 66.93 55.35 62.00 8.02 17.28 19.0420.13 7.89 4.91 11.21 5.35 4 8.58 1.86 1.68 8.45 13.79 10.61 12.84 8.2765.06 51.89 61.94 8.06 19.29 18.85 25.22 7.96 6.28 4.91 5 8.60 1.35 1.781.45 8.45 12.35 13.63 8.59 8.27 60.12 55.07 64.28 8.07 20.50 20.35 19.737.94 5.69 9.17 5.95 6 8.55 1.30 1.30 1.08 8.43 13.29 13.26 13.23 8.2454.83 56.88 61.67 8.08 20.76 19.79 17.19 7.96 9.82 8.76 6.84 7 8.57 1.281.41 2.40 8.44 12.08 12.63 13.05 8.27 61.50 55.33 60.70 8.08 17.55 19.4817.43 7.94 5.93 8.92 4.25 8 8.55 1.32 0.90 8.43 11.51 12.47 10.09 8.2462.99 54.09 63.81 8.05 15.43 22.71 20.91 7.90 8.75 9.83 5.19 9 8.59 1.351.63 8.45 11.59 13.67 11.40 8.28 63.60 52.70 63.11 8.06 17.98 24.0818.57 7.94 2.28 7.05 5.29 10 8.57 1.56 2.50 2.08 8.45 13.22 11.93 12.908.28 61.86 55.12 61.87 8.08 17.87 20.99 18.74 7.97 5.50 4.71 4.41 118.59 1.43 1.19 8.45 12.25 11.42 9.85 8.28 58.83 59.88 64.13 8.08 18.1822.06 17.46 7.97 9.61 5.45 6.97 12 8.56 1.64 1.39 0.94 8.39 15.30 13.0715.71 8.21 63.76 59.71 62.92 8.02 16.72 20.51 16.60 7.97 2.58 4.21 3.85

Results of Block F

In this block of formulations, the pH values were all slightly lowerthan the target value of pH 5.2 (Table F-1). In addition, the pH doeschange by about 0.1 units for most of the formulations when measured att1. These differences were considered when constructing mathematicalmodels of the data, as discussed below.

The addition of EDTA does appear to improve stability for the worstformulation (Formulation 1). Whether it increases stability in generalwas less clear, based on the SEC data (Table F-2). The formulationscontaining high concentrations of Arg or Gly all performed quite wellupon storage (Table F-2).

The initial purities by RP HPLC were universally lower than expected forthese formulations (Table F-3). Upon storage at t1 and t2, there aresome slight differences, with Gly- and Arg-based formulations showingthe greatest stability. Based upon the RP HPLC data, EDTA does notappear to be a significant stabilizer (Table F-3). Likewise, the effectof Met appears to be minimal on stability as measured by RP HPLC or SEC,with the exception of the monomer content for the highest Metconcentration (Table F-2, Formulation 7).

Analysis by CE-SDS indicates that very little degradation occurs uponstorage (usually less than 1% increase in ‘Other’) (Table F-4). However,there are some formulations that begin with higher ‘Other’ contents(Formulations 4 through 7, for example). These are all formulationsusing a high concentration of mannitol (240 mM). The same seems to betrue for formulations containing 120 mM mannitol.

As for analysis by cIEF, there is little change in the relativeintensities of the main peak, at least in a systematic fashion thatwould allow one to discern stability trends (Table F-5). In general, thechanges are smaller at t2 than at t1.

Block G Formulation Studies

The Block G formulation studies examined a variety of formulations withcombinations of Gly and Arg as the primary stabilizers (Table XXXIV). Inaddition, two other surfactants (Pluronic F-68 and polysorbate 20, PS20) were evaluated in addition to PS 80. Finally, a range of PS 80concentrations was evaluated.

TABLE G BLOCK G STUDY DESIGN Form No. API citrate phosphate succinateHIS Gly Arg mannitol NaCl F68 PS20 PS80 1 Adalimumab 8 18 0 0 0 0 65 1000 0 0.1 biosimilar 2 Adalimumab 8 18 0 0 0 0 65 100 0 0.1 0 biosimilar 3Adalimumab 8 18 0 0 0 0 65 100 0.1 0 0 biosimilar 4 Adalimumab 0 0 0 10120 120 0 0 0 0 0.1 biosimilar 5 Adalimumab 0 0 0 10 120 120 0 0 0 00.05 biosimilar 6 Adalimumab 0 0 0 10 120 120 0 0 0 0 0.01 biosimilar 7Adalimumab 0 0 0 10 120 120 0 0 0 0.05 0 biosimilar 8 Adalimumab 0 0 010 120 120 0 0 0.1 0 0 biosimilar 9 Adalimumab 0 0 10 0 120 120 0 0 0 00.05 biosimilar 10 Adalimumab 0 0 20 0 150 100 0 0 0 0.05 0 biosimilar11 Adalimumab 0 0 0 20 150 100 0 0 0 0 0.01 biosimilar 12 Adalimumab 0 00 20 120 120 0 0 0 0.01 0 biosimilar

TABLE G-1 Measured pH for Block G formulations at t0, t1 (one week, 40°C.), and t2 (two weeks, 40° C.) Form No. citrate phosphate succinate HisGly Arg mannitol NaCl F68 PS20 PS80 t0 t1 t2 1 8 18 0 0 0 0 65 100 0 00.1 5.19 5.38 5.25 2 8 18 0 0 0 0 65 100 0 0.1 0 5.23 5.28 5.24 3 8 18 00 0 0 65 100 0.1 0 0 5.22 5.26 5.20 4 0 0 0 10 120 120 0 0 0 0 0.1 5.205.33 5.29 5 0 0 0 10 120 120 0 0 0 0 0.05 5.23 5.34 5.29 6 0 0 0 10 120120 0 0 0 0 0.01 5.19 5.40 5.27 7 0 0 0 10 120 120 0 0 0 0.05 0 5.235.39 5.42 8 0 0 0 10 120 120 0 0 0.1 0 0 5.19 5.38 5.41 9 0 0 10 0 120120 0 0 0 0 0.05 5.19 5.27 5.24 10 0 0 20 0 150 100 0 0 0 0.05 0 5.235.28 5.24 11 0 0 0 20 150 100 0 0 0 0 0.01 5.23 5.33 5.27 12 0 0 0 20120 120 0 0 0 0.01 0 5.22 5.29 5.29

TABLE G-2 Monomer content by SEC for formulations in Block G at t0, t1(one week at 40° C.), and t2 (two weeks at 25° C.) Form No. citratephosphate succinate His Gly Arg mannitol NaCl F68 PS20 PS80 t0 t1 t2 1 818 0 0 0 0 65 100 0 0 0.1 99.17 97.45 98.09 2 8 18 0 0 0 0 65 100 0 0.10 99.11 97.78 98.09 3 8 18 0 0 0 0 65 100 0.1 0 0 98.99 97.74 97.92 4 00 0 10 120 120 0 0 0 0 0.1 99.12 98.67 98.68 5 0 0 0 10 120 120 0 0 0 00.05 99.05 98.57 98.53 6 0 0 0 10 120 120 0 0 0 0 0.01 99.05 98.66 98.707 0 0 0 10 120 120 0 0 0 0.05 0 99.04 98.63 98.50 8 0 0 0 10 120 120 0 00.1 0 0 99.11 98.64 98.55 9 0 0 10 0 120 120 0 0 0 0 0.05 99.12 98.5698.98 10 0 0 20 0 150 100 0 0 0 0.05 0 99.10 98.49 98.88 11 0 0 0 20 150100 0 0 0 0 0.01 99.07 98.76 98.45 12 0 0 0 20 120 120 0 0 0 0.01 099.11 98.48

TABLE G-3 Percent purity by RP HPLC for formulations in Block G at t0,t1 (one week at 40° C.), and t2 (two weeks at 25° C. Form No. citratephosphate succinate HIS Gly Arg mannitol NaCl F68 PS20 PS80 t0 t1 t2 1 818 0 0 0 0 65 100 0 0 0.1 99.74 99.66 98.93 2 8 18 0 0 0 0 65 100 0 0.10 99.59 99.66 98.97 3 8 18 0 0 0 0 65 100 0.1 0 0 99.58 99.60 99.22 4 00 0 10 120 120 0 0 0 0 0.1 99.62 99.62 98.99 5 0 0 0 10 120 120 0 0 0 00.05 99.70 99.61 99.01 6 0 0 0 10 120 120 0 0 0 0 0.01 99.60 99.66 99.007 0 0 0 10 120 120 0 0 0 0.05 0 99.71 99.65 98.99 8 0 0 0 10 120 120 0 00.1 0 0 99.70 99.61 99.03 9 0 0 10 0 120 120 0 0 0 0 0.05 99.71 99.6099.03 10 0 0 20 0 150 100 0 0 0 0.05 0 99.72 99.60 99.02 11 0 0 0 20 150100 0 0 0 0 0.01 99.72 99.61 99.05 12 0 0 0 20 120 120 0 0 0 0.01 099.61 99.04

TABLE G-4 Percentage of bands for light chain (LC), heavy chain (HC),non- glycosylated HC, and other species for formulations in Block G att0, t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. TimeLC HC ngHC Other 1 t0 28.55 70.77 0.50 0.17 t1 29.71 69.42 0.57 0.30 t230.32 68.80 0.53 0.35 2 t0 37.14 62.38 0.49 0.00 t1 30.31 69.38 0.280.03 t2 31.60 67.87 0.53 0.00 3 t0 28.95 70.40 0.65 0.00 t1 28.17 70.260.58 0.99 t2 27.32 71.52 0.56 0.59 4 t0 29.56 69.02 0.65 0.77 t1 32.1966.09 0.53 1.19 t2 31.58 66.03 0.57 1.81 5 t0 36.54 62.48 0.56 0.42 t128.77 69.28 0.62 1.33 t2 23.76 74.49 0.60 1.16 6 t0 29.60 68.61 0.581.21 t1 30.37 67.42 0.59 1.61 t2 32.27 66.08 0.59 1.06 7 t0 31.90 65.500.63 1.97 t1 31.26 66.66 0.56 1.51 t2 31.37 66.64 0.67 1.31 8 t0 31.0467.38 0.54 1.04 t1 30.34 67.99 0.62 1.05 t2 30.21 67.63 0.68 1.48 9 t033.12 65.34 0.61 0.94 t1 34.01 63.97 0.56 1.46 t2 34.47 63.77 0.57 1.1910 t0 36.78 61.61 0.54 1.07 t1 39.25 58.66 0.53 1.56 t2 32.83 65.42 0.551.21 11 t0 36.37 61.97 0.54 1.11 t1 t2 34.97 63.14 0.54 1.36 12 t0 34.2664.16 0.52 1.05 t1 t2 34.90 63.35 0.56 1.19

TABLE G-5 Percentage of main bands seen in the cIEF profile offormulations in Block G at t0, t1 (one week at 40° C.), and t2 (twoweeks at 25° C. Form No. pH t0 t1 t2 1 8.53 1.24 1.21 8.36 14.30 12.6913.67 8.24 64.03 53.50 60.30 8.14 8.01 15.77 9.32 19.12 7.86 3.73 3.354.48 2 8.52 1.06 1.37 0.88 8.35 13.10 13.30 12.53 8.16 66.28 59.68 57.997.97 17.14 19.60 21.55 7.83 2.42 4.78 4.92 3 8.51 0.65 0.65 1.03 8.3413.31 14.00 15.31 8.16 65.13 59.04 60.70 8.14 7.98 17.26 18.90 17.567.82 2.89 5.68 4.16 4 8.36 1.87 2.43 1.00 8.19 7.74 10.89 11.69 7.9961.91 54.27 59.10 7.82 20.94 22.72 19.81 7.66 6.35 7.98 6.92 5 8.44 1.790.95 071 8.26 13.33 12.85 10.43 8.06 61.67 59.94 60.12 7.88 17.49 21.0820.82 7.69 4.02 4.50 6.50 6 8.36 1.71 4.76 8.21 12.37 12.93 10.95 8.0462.53 54.16 56.48 7.87 19.24 26.08 17.97 7.64 4.15 2.07 6.50 7 8.54 0.771.19 0.79 8.34 7.15 12.32 13.15 8.17 54.73 42.64 60.58 8.02 22.18 29.9017.28 7.83 7.12 11.47 4.77 7.69 1.41 2.48 2.11 8 8.55 1.04 2.11 8.397.28 10.69 14.82 8.23 64.01 57.42 55.68 8.05 20.81 23.86 23.76 7.96 6.795.37 5.74 9 8.54 8.48 10.99 7.91 8.31 53.85 61.43 8.17 31.58 23.83 7.998.82 7.85 3.58 3.27 10 8.50 0.95 2.16 8.36 9.10 10.65 15.79 8.18 59.0255.35 58.56 8.02 23.76 24.79 25.66 7.87 5.63 7.05 11 8.58 2.08 1.68 8.409.74 10.05 9.67 8.21 62.70 56.96 57.36 8.05 21.39 24.14 25.18 7.99 5.246.77 6.11 12 8.54 1.67 8.37 15.99 8.22 63.18 8.02 15.41 7.82 3.75

TABLE G-6 Block G study design for F/T and agitation studies Form No.API citrate phosphate succinate HIS Gly Arg mannitol NaCl F68 PS20 PS801 Adalimumab 8 18 0 0 0 0 65 100 0 0 0.1 biolsimilar 2 Adalimumab 8 18 00 0 0 65 100 0 0.1 0 biolsimilar 3 Adalimumab 8 18 0 0 0 0 65 100 0.1 00 biolsimilar 4 Adalimumab 0 0 0 10 120 120 0 0 0 0 0.1 biolsimilar 5Adalimumab 0 0 0 10 120 120 0 0 0 0 0.05 biolsimilar 6 Adalimumab 0 0 010 120 120 0 0 0 0 0.01 biolsimilar 7 Adalimumab 0 0 0 10 120 120 0 0 00.05 0 biolsimilar 8 Adalimumab 0 0 0 10 120 120 0 0 0.1 0 0 biolsimilar9 Adalimumab 0 0 10 0 120 120 0 0 0 0 0.05 biolsimilar 10 Adalimumab 0 020 0 150 100 0 0 0 0.05 0 biolsimilar 11 Adalimumab 0 0 0 20 150 100 0 00 0 0.01 biolsimilar 12 Adalimumab 0 0 0 20 120 120 0 0 0 0.01 0biolsimilar

TABLE G-7 Monomer content by SEC for select formulations in Block G thatwere untreated (Q, quiescent), underwent 5 F/T cycles or subjected toagitation for 24 hours Form No. citrate phosphate succinate HIS Gly Argmannitol NaCl F68 PS20 PS80 Q F/T agit 1 8 18 0 0 0 0 65 100 0 0 0.199.15 99.03 99.14 4 0 0 0 10 120 120 0 0 0 0 0.1 99.21 99.11 99.18 8 0 00 10 120 120 0 0 0.1 0 0 99.18 99.14 99.17 11 0 0 0 20 150 100 0 0 0 00.01 99.16 99.09 99.13 12 0 0 0 20 120 120 0 0 0 0.01 0 99.10

TABLE G-8 Percent purity by RP HPLC for select formulations in Block Gthat were untreated (Q, quiescent), underwent 5 F/T cycles or subjectedto agitation for 24 hours Form No. citrate phosphate succinate HIS GlyArg mannitol NaCl F68 PS20 PS80 Q F/T agit 1 8 18 0 0 0 0 65 100 0 0 0.199.60 99.72 99.76 4 0 0 0 10 120 120 0 0 0 0 0.1 99.56 99.70 99.59 8 0 00 10 120 120 0 0 0.1 0 0 99.58 99.57 99.73 11 0 0 0 20 150 100 0 0 0 00.01 99.72 99.59 99.65 12 0 0 0 20 120 120 0 0 0 0.01 0 99.75 99.56

Results of Block G

All of the pH values were close to the target values (Table G-1), withrelatively small changes occurring upon storage. There appears to besome preference in terms of polysorbates over F-68 in terms ofstability, as measured by SEC (Table G-2). However, the differences arerelatively small. It does appear that succinate formulations(Formulations 9 and 10) fared reasonably well as far as monomer contentretained.

The RP HPLC data are all very close, making any determination ofstability differences virtually impossible (Table G-3). These data willonly be interpretable when examined in the larger context of all of theblocks of screening studies.

The CE-SDS results suggest that PS 20 is the best stabilizer at 0.1%concentration for the Humira® formulation (Formulations 1 through 3)(Table G-4). Otherwise, the differences appear to be too small andvariable to make any general conclusions.

As seen before, the results for cIEF data are variable enough to makeinterpretation difficult (Table G-5). It does appear that the changesare smaller in the Gly/Arg formulations than for formulations usingother stabilizers, like mannitol. Still, overall, the stability by cIEFlooks to be quite good for many of the formulations in this study.

Block G (F/T and Agitation) Studies.

For a liquid formulation, it is important to evaluate the sensitivity tointerfacial stress. Two kinds of stress studies were selected. The firstis agitation at 150 rpm on an orbital shaker for 24 hours at 2-8° C. Thesecond is five successive cycles of freezing and thawing (F/T), wherethis cycle should generate increasing amounts of damage protein, if theprotein is sensitive to interfacial damage. Four formulations from BlockG were selected for assessment, and they are highlighted in blue boldtext Table G-6.

Upon repeated F/T cycling, there is a very small decrease in monomercontent for all of the formulations tested (Table G-7). Thus, it seemslike there is little interfacial sensitivity form this stress and thatthe presence of PS 80 is not critical for protection. As for agitatedsamples, the losses are even smaller. The trends in the RP HPLC data areessentially the same (Table G-8). There are little, if any, losses inpurity upon exposure to interfacial stress.

Block H Formulation Studies

The Block H formulations focused on three aspects of the adalimumabformulation: (1) higher protein concentrations, (2) formulations with nobuffers present (other than the protein), and (3) the use of variousbuffer combinations beside citrate-phosphate (See Table H).

TABLE H BLOCK H STUDY DESIGN Form No. API protein citrate phosphatesuccinate HIS ACETATE Gly Arg mannitol NaCl PS80 1 *** 100 8 18 0 0 0 00 65 100 0.1 2 *** 100 0 0 0 10 0 120 120 0 0 0.1 3 *** 50 0 0 0 0 0 0 065 100 0.1 4 *** 50 0 0 0 0 0 120 120 0 0 0.1 5 *** 50 0 0 0 0 0 120 1200 0 0 6 *** 50 0 0 0 10 10 0 0 65 100 0.1 7 *** 50 0 0 10 10 0 0 0 65100 0.1 8 *** 50 0 10 0 10 0 0 0 65 100 0.1 9 *** 50 0 0 10 0 10 0 0 65100 0.1 10 *** 50 10 0 10 0 0 0 0 65 100 0.1 11 *** 50 10 0 0 10 0 0 065 100 0.1 12 *** 50 0 0 10 10 0 120 100 0 0 0.1 *** denotes proprietaryadalimumab biosimilar

TABLE H-1 Measured pH for Block H formulations at t0, t1 (one week, 40°C.), and t2 (two weeks, 40° C.) Form No. protein Citrate PhosphateSuccinate Histidine acetate Gly Arg Mannitol NaCl PS80 t0 t1 t2 1 100 818 0 0 0 0 0 65 100 0.1 5.19 5.30 5.29 2 100 0 0 0 10 0 120 120 0 0 0.15.20 5.19 5.15 3 50 0 0 0 0 0 0 0 65 100 0.1 5.21 5.23 5.21 4 50 0 0 0 00 120 120 0 0 0.1 5.21 5.41 5.46 5 50 0 0 0 0 0 120 120 0 0 0 5.21 5.305.39 6 50 0 0 0 10 10 0 0 65 100 0.1 5.20 5.28 5.28 7 50 0 0 10 10 0 0 065 100 0.1 5.21 5.24 5.24 8 50 0 10 0 10 0 0 0 65 100 0.1 5.20 5.17 5.169 50 0 0 10 0 10 0 0 65 100 0.1 5.21 5.24 5.29 10 50 10 0 10 0 0 0 0 65100 0.1 5.20 5.24 5.26 11 50 10 0 0 10 0 0 0 65 100 0.1 5.21 5.24 5.2612 50 0 0 10 10 0 120 100 0 0 0.1 5.21 5.26 5.29

TABLE H-2 Monomer content by SEC for formulations in Block H at t0, t1(one week at 40° C.), and t2 (two weeks at 25° C.) Form No. proteinCitrate Phosphate Succinate Histidine acetate Gly Arg mannitol NaCl PS80t0 t1 t2 1 100 8 18 0 0 0 0 0 65 100 0.1 99.25 98.36 98.42 2 100 0 0 010 0 120 120 0 0 0.1 99.19 98.88 98.47 3 50 0 0 0 0 0 0 0 65 100 0.199.06 98.81 98.74 4 50 0 0 0 0 0 120 120 0 0 0.1 99.19 99.06 98.99 5 500 0 0 0 0 120 120 0 0 0 99.26 99.03 98.96 6 50 0 0 0 10 10 0 0 65 1000.1 99.26 98.92 98.86 7 50 0 0 10 10 0 0 0 65 100 0.1 99.14 98.98 98.938 50 0 10 0 10 0 0 0 65 100 0.1 99.11 98.93 98.66 9 50 0 0 10 0 10 0 065 100 0.1 99.16 98.79 98.63 10 50 10 0 10 0 0 0 0 65 100 0.1 99.1098.79 98.49 11 50 10 0 0 10 0 0 0 65 100 0.1 99.21 98.93 98.18 12 50 0 010 10 0 120 100 0 0 0.1 99.30 99.22 98.65

TABLE H-3 Percent purity by RP HPLC for formulations in Block F at t0,t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. proteincitrate phosphate succinate histidine acetate Gly Arg mannitol NaCl PS80t0 t1 t2 1 100 8 18 0 0 0 0 0 65 100 0.1 99.36 99.64 99.64 2 100 0 0 010 0 120 120 0 0 0.1 99.37 99.68 99.74 3 50 0 0 0 0 0 0 0 65 100 0.199.45 99.47 99.70 4 50 0 0 0 0 0 120 120 0 0 0.1 99.50 99.69 99.59 5 500 0 0 0 0 120 120 0 0 0 99.47 99.71 99.56 6 50 0 0 0 10 10 0 0 65 1000.1 99.48 99.56 99.72 7 50 0 0 10 10 0 0 0 65 100 0.1 99.43 99.45 99.728 50 0 10 0 10 0 0 0 65 100 0.1 99.43 99.51 99.72 9 50 0 0 10 0 10 0 065 100 0.1 99.47 99.55 99.72 10 50 10 0 10 0 0 0 0 65 100 0.1 99.4899.53 99.67 11 50 10 0 0 10 0 0 0 65 100 0.1 99.45 99.69 99.60 12 50 0 010 10 0 120 100 0 0 0.1 99.44 99.54 99.72

TABLE H-4 Percentage of bands for light chain (LC), heavy chain (HC),non- glycosylated HC, and other species for formulations in Block H att0, t1 (one week at 40° C.), and t2 (two weeks at 25° C.) Form No. TimeLC HC ngHC Other 1 t0 32.87 65.48 0.54 1.11 t1 28.08 70.09 0.58 1.25 t252.57 47.43 0.00 0.00 2 t0 36.20 62.40 0.55 0.86 t1 29.64 68.68 0.571.11 t2 43.09 55.23 0.57 1.10 3 t0 34.70 63.55 0.57 1.18 t1 28.24 69.720.61 1.57 t2 34.25 63.97 0.67 1.11 4 t0 41.04 57.61 0.51 0.84 t1 27.5870.65 0.62 1.15 t2 34.14 64.01 0.60 1.26 5 t0 37.64 60.77 0.50 1.09 t128.07 70.02 0.61 1.30 t2 37.67 60.76 0.55 1.02 6 t0 31.64 66.46 0.551.34 t1 27.67 70.19 0.50 1.64 t2 34.07 63.49 0.62 1.81 7 t0 30.38 69.100.53 0.00 t1 27.14 70.55 0.62 1.69 t2 46.41 51.21 0.00 2.38 8 t0 28.4671.19 0.35 0.00 t1 30.05 68.71 0.55 0.69 t2 34.14 63.97 0.63 1.25 9 t027.74 70.63 0.60 1.03 t1 27.48 70.48 0.61 1.43 t2 36.56 61.59 0.49 1.3610 t0 t1 27.69 70.46 0.60 1.24 t2 11 t0 27.64 70.83 0.57 1.13 t1 31.8566.08 0.61 1.46 t2 38.58 59.26 0.52 1.64 12 t0 29.48 68.55 0.58 1.40 t129.53 68.68 0.58 1.40 t2 30.64 68.20 0.70 0.46

TABLE H-5 Percentage of main bands seen in the cIEF profile offormulations in Block H at t0, t1 (one week at 40° C.), and t2 (twoweeks at 25° C.) Form No. pH t0 t1 t2 1 8.55 1.20 1.17 1.21 8.39 9.575.57 8.23 8.23 46.84 38.18 39.78 7.99 13.67 12.64 11.62 7.81 6.93 4.613.70 2 8.43 1.17 1.06 1.38 8.26 8.97 8.15 8.38 8.09 45.46 40.27 39.957.87 13.37 16.45 6.77 7.72 5.47 5.39 9.55 7.56 1.64 1.52 3.33 3 8.360.80 0.74 0.61 8.16 6.02 6.03 7.30 7.98 35.60 35.58 37.24 7.83 11.7514.10 13.15 7.64 2.17 4.78 2.00 7.51 1.23 1.81 4 8.40 0.82 0.74 0.308.22 7.87 7.38 6.29 8.04 42.46 34.42 35.89 7.89 14.44 13.71 11.34 7.713.18 3.31 2.69 7.56 0.98 0.95 5 8.42 0.82 1.02 8.25 7.22 5.09 8.07 34.6828.99 7.91 2.67 3.63 7.86 10.63 7.83 7.72 2.52 2.05 6 8.42 1.17 1.281.22 8.23 9.88 8.56 7.90 8.09 45.26 40.45 40.80 7.94 13.23 16.50 13.28 78.59 1.79 1.45 1.90 8.45 11.74 11.32 11.51 8.28 59.90 61.63 56.22 8.0520.34 19.49 22.98 7.92 6.24 6.11 7.39 8 8.58 1.59 2.94 1.38 8.44 11.8612.83 12.12 8.26 61.08 60.20 63.05 8.05 20.21 24.03 23.45 7.88 5.25 6.559 8.61 1.22 1.42 1.21 8.48 12.47 12.36 11.00 8.33 56.64 54.59 55.34 8.1023.37 23.81 25.31 7.94 6.30 7.83

Results of Block H

The pH stability of these formulations was acceptable (<0.1 units),except for Formulations 4 and 5. These are the buffer-free formulationsusing Gly and Arg as the stabilizers (Table H-1). There was also aslight rise in pH for Formulation 1 (the Humira® formulation at 100mg/ml protein concentration).

Stability of Block H formulations was monitored using SEC and RP HPLC.There is little loss in monomer content, with Formulation 1 appearing tobe the least stable by SEC (Table H-2). At 100 mg/ml of adalimumabbiosimilar API the histidine-buffered formulation containing Gly and Argappears to be quite stable. In general, the best buffer combinationappears to be His-succinate (Formulations 7 and 12). Buffer-freeformulations with Gly and Arg show acceptable stability as well (TableH-2). The RP HPLC data indicate that the buffer-free formulations (4 and5) may not do quite as well as shown by SEC (Table H-3), with measurabledecreases in purity, but are believed to be satisfactory for obtaining aformulation having long term stability.

The CE-SDS data detect the least change in Formulation 12, which is aHis-succinate formulation (Table H-4). The largest change at t1 occurswith Formulation 7, which is also a His-succinate formulation, but usingmannitol and NaCl as the tonicity modifiers.

PLS Modeling PLS Method

The data for the adalimumab formulations in Blocks A through H wereanalyzed together using a chemometric method termed partial leastsquares (PLS).

Detailed descriptions of PLS modeling have been published. See, forexample, Katz, M. H. Multivariate Analysis: A Practice Guide forClinicians. Cambridge University Press, New York, pp. 158-162 (1999);Stahle, L., Wold, K., Multivariate data analysis and experimental designin biomedical research. Prog. Med. Chem. 1988, 25: 291-338; Wold S.PLS-regression: a basic tool of chemometrics. Chemom. Intell. Lab. Syst.2001, 58: 109-130; and Martens, H.; Martens, M. Multivariate Analysis ofQuality: An Introduction, Wiley and Sons, Chichester, UK (2001).

For any large matrix of values, where there are a reasonable number ofsamples (together forming the so-called X-matrix), mathematical modelscan be constructed that explain the largest amount of variance in thedependent variable(s) of interest (the Y-matrix). The best singledescription of the relationship between the variation in the X-matrixand the endpoint (the Y matrix) is called the first principal component,PC1. The next important (in terms of describing the variance in theY-matrix) component is called the second principal component, PC2, andso on. Quite often, only one or two PCs are required to explain most ofthe variance in the Y-matrix. Each of these PCs contains somecontribution from each of the variables in the X-matrix. If a variablewithin the X-matrix contributes heavily to the construction of a givenPC, then it is ranked as being significant. In fact, regressioncoefficients can be calculated for each variable in the X-matrix for agiven model, where a model is the composite of a certain number of PCsin order to provide an adequate description of the Y-matrix. In summary,PLS takes information from the X-matrix, calculates the desired numberof PCs, and constructs a suitable model. The model that includes all ofthe samples is termed a calibration model [1,2]. The overall coefficientof determination (r²) indicates the quality of the model. All PLScalculations were conducted using Unscrambler® software (CAMO,Corvallis, Oreg.). A PLS analysis done with a single variable in theY-matrix is termed PLS1 analysis. Building a model that fits multiplevariables in the Y-matrix is called PLS2 analysis.

A full cross validation was performed on all calibration models usingstandard techniques. Briefly, one sample is removed at a time, the dataset is recalibrated, and a new model is constructed. This process isrepeated until all of the calibration samples are removed once andquantified as a validation model. Therefore, the first set, containingall samples is referred to as the calibration set and the one aftercross-validation as the validation set. The jack-knife algorithm (See,Martens et al) was used to determine statistical significance for anyfactor used in constructing the PLS models described above.

PLS Modeling of Adalimumab Formulations BLOCKS B, C and D See FIGS. 3Through 12

Note: The PLS surface graphs depicted in FIGS. 3 through 12 are based onthe data obtained from Blocks B, C and D. The following is a discussionof the findings reflected in the PLS surface plots shown in FIGS. 3through 12.

PLS Model 1—FIG. 3.

FIG. 3 contains a depiction of the monomer content at t1 (model 1) as afunction of citrate and phosphate concentrations. The pH has been fixedat 5.2. The model indicated that phosphate and citrate by themselveswere weak destabilizers (not to statistical significance), along withtartrate and maleate. By comparison, succinate, which is structurallysimilar to citrate, tartrate and maleate, was a weak stabilizer. Theonly buffer found to be a significant stabilizer was histidine. None ofthese findings could have been predicted based on the literature orexamination of the chemical structure of each buffer. The model alsoindicated that when citrate and phosphate buffer are used together, theformulation is least stable. If one only uses a single buffer,especially phosphate, stability improves. This is surprising, asphosphate has little or no buffer capacity at pH 5.2, while citratebuffer does. None of this behavior could have been predicted based onwhat was known in the art.

PLS Model 2—FIG. 4.

FIG. 4 contains a depiction of the monomer content at t2 (model 2).Likewise, a model was constructed using the monomer content by SEC at t2as the endpoint. This model also demonstrated that the stability islowest when citrate and phosphate are used together. The loweststability was shown when citrate is above 10 mM and phosphate is between5 and 15 mM. Stability improves when the citrate concentration islowered and/or phosphate concentration is lowered or raised. Thesefindings suggest that a single buffer composition is preferred. The sametrend in buffer stabilization is seen as with PLS Model 1, with citrateand phosphate being weak stabilizers (not statistically significant),while histidine is a strong stabilizer (statistically significant).

PLS Model 1—FIG. 5.

FIG. 5 is a PLS model 1 showing the effect of histidine and glycine onthe stability of formulations. It contains a depiction of the monomercontent at t1 (model 1). This model indicated that the combination ofhistidine and glycine yielded very good stability results. Bothhistidine (His) and glycine (Gly) were determined to be stabilizers. Thelowest stability on the response surface (shown in blue) is when thereis the lowest concentration of His and Gly. The effect of His onstability is greater, with 20 mM His providing comparable stabilizationto 120 mM Gly (note the opposite corners of the graph). The modelindicates that there will be an additive benefit to stability by usingboth excipients, with the highest stability occurring when the Hisconcentration is 20 mM and the Gly concentration is 120 mM.

PLS Model 1—FIG. 6.

FIG. 6 is a PLS model 1 showing the effect of arginine and sorbitol onthe stability of formulations. It contains a depiction of the monomercontent at t1 (model 1). This model indicated that arginine was a goodstabilizer, while sorbitol was a poor stabilizer. Likewise, arginine(Arg) provides a degree of stabilization that is similar to that foundfor Gly. The poorest stability as indicated by this model is when theArg concentration is low and the sorbitol concentration is low (the bluearea of the graph). As the concentrations of each excipient areincreased, the monomer content at t1 is increased. The effect ofsorbitol is roughly linear with concentration, while the effect of Argappears to be increasing more rapidly once the concentration exceeds 60mM. Even though sorbitol is predicted to increase the stability ofadalimumab in terms of retained monomer content, its ability to increasestability is less than that found for Gly and Arg (on a molar basis).

PLS Model 1—FIG. 7.

FIG. 7 is a PLS model 1 showing the effect of pH and histidine on thestability of formulations. It contains a depiction of the monomercontent at t1 (model 1). This model indicated that histidine appears tobe the best buffer, while pH should preferably be at 5 or higher forbest stability.

PLS Model 2—FIG. 8.

FIG. 8 is a PLS model 2 showing the effect of pH and histidine on thestability of formulations. It contains a depiction of the monomercontent at t2 (model 2). This model indicated that histidine appears tobe the best buffer, while pH should preferably be at 5 or higher forbest stability. The results indicate that the optimal pH is near 5.2. Ofall of the buffers that were examined, histidine provides the greatestdegree of stabilization. This response surface illustrates two importantpoints. First, the stability appears to be maximal near pH 5.2, fallingoff at a higher and lower pH. Second, histidine is shown to provide asignificant increase in stability. When histidine is used at 20 mM, itprovides a marked increase in stability over lower bufferconcentrations. In fact, the effect appears to be non-linear, with morestabilization occurring from 10 to 20 mM than from 0 to 10 mM. Further,the loss in stability is more abrupt at higher pH than at lower pH.

PLS Model 2—FIG. 9.

FIG. 9 is a PLS model 2 showing the effect of trehalose and PS80 on thestability of formulations. It contains a depiction of the monomercontent at t2. This model indicated that trehalose appears to be a weakstabilizer, while PS80 improves thermal stability. The response surfaceshown in FIG. 9 indicates that PS 80 is a potent stabilizer forprotecting adalimumab against thermal stress, with a concentration of0.1% providing maximal stability. The concentration of PS 80 has notbeen varied other than at 0 and 0.1%. By comparison, this model showsthat the stabilization effect of trehalose is quite small, certainlyless than what was seen with sorbitol.

PLS Model 2—FIG. 10.

FIG. 10 is a PLS model 2 showing the effect of mannitol and PS80 on thestability of formulations. It contains a depiction of the monomercontent at t2 (model 2). This model indicated that mannitol appears tobe a destabilizer, while PS80 improves thermal stability. The PLS modelusing monomer content by SEC at t2 allows one to examine the relativeeffects of any of the factors included in the model. As the mannitolconcentration increases, the overall stability decreases. By comparison,the impact of PS80 on the stability is rather small.

PLS Model 1—FIG. 11.

FIG. 11 is a PLS model 1 showing the effect of mannitol and NaCl on thestability of formulations. It contains a depiction of the monomercontent at t1 (model 1). This model indicated that mannitol and NaClboth appear to be destabilizers. The stability, as indicated by themonomer content at t1, is lowest when the mannitol concentration isanywhere below 150 mM. Likewise, addition of NaCl also diminishes thestability of adalimumab.

PLS Model 1—FIG. 12.

FIG. 12 is a PLS model 1 showing the effect of EDTA and methionine onthe stability of formulations. It contains a depiction of the monomercontent at t1. In the case of EDTA, the stability decreases slightly asthe concentration of this additive increases. In contrast, addition, ofMet appears to improve stability.

PLS Modeling of Adalimumab Formulations for Blocks B Through G See FIGS.13 Through 28 The First PLS Model (“PLS Model A)

The first PLS model (PLS Model A) used difference in monomer content att1 as the endpoint. The model employed three PCs and had a correlationcoefficient for the calibration set of 0.83 and a r-value of 0.67 forthe validation set. It was a quadratic model including pH-buffer andbuffer-buffer interaction terms.

TABLE J PLS “MODEL A” COEFFICIENTS Factor r-value pH t0 0.041 protein−0.025 citrate +0.123 phos +0.267 succinate −0.089 histidine −0.174acetate −0.053 glycine −0.190 arginine −0.128 sorbitol −0.003 trehalose+0.020 mannitol −0.104 NaCl +0.250 F68 +0.018 PS 20 +0.021 PS 80 −0.152EDTA +0.112 Met −0.062 Note: Overall correlation coefficients for eachlinear factor includes in the first PLS model (PLS Model A) using thedifference in monomer content by SEC at t1 as the endpoint. Factorsdeemed to be statistically significant are highlighted in bold text.

The model quality is acceptable, considering the correlationcoefficients of the calibration and validation sets. The overallcorrelation coefficients for the various factors included in the modelare summarized in Table J. Note that the quadratic and interaction termsare not listed here. As the endpoint is the difference in monomercontent, one wishes to minimize this value. Thus, stabilizers exhibitnegative correlation coefficients, while destabilizers have positiver-values. Of the stabilizers, His, Gly, Arg, and PS 80 are the mostpotent, although mannitol and succinate also have a stabilizing effect(Table J). Meanwhile, there are some significant destabilizers, such asNaCl, citrate, and phosphate. Keep in mind that these models are acomposite of all of the stability data gathered across the variousblocks of formulations, A through H, and individual formulations couldvary from the model. While the table of correlation coefficients ishelpful to gauge the effects of various factors, they do not capture thenon-linear and interaction effects, so it is helpful to view responsesurfaces to examine the effects of various parameters in greater detail,as shown in the response surfaces that are reproduced in FIGS. 13through 28.

Discussion of PLS Model A—FIGS. 13 and 14.

The Krause '583 patent describes the citrate-phosphate buffer system asbeing integral to product stability. Our studies show this not to be thecase. The poorest stability would occur when these two buffers were usedin combination and the effect would get worse as the bufferconcentrations increase, according to this model (FIG. 13[1]). Theresponse surface indicates that the phosphate and citrate are equallydestabilizing, contrary to some earlier observations, but thequantitative nature of these surfaces must be considered with some careas they include data from all of the formulations from Blocks B throughH.

The effect of pH and His is shown in FIG. 14. It shows that His isdestabilizing at low pH, where it is clearly outside of the buffercapacity of His. Again, this result is a function of all pH observationsin this study, not just those involving His (although this could bedone). According to this response surface, the optimal pH may be nearerto 5.4 than 5.2, although the surface is relatively flat through thisregion, indicating a shallow response surface from pH 5 to 5.4 (FIG.14).

Discussion of PLS Model A—FIG. 15

The response surface for Gly and Arg is shown in FIG. 15. The studiesrepeatedly show that these two amino acids can be potent stabilizers ofadalimumab. Note that the minimum difference in monomer content (i.e.,the blue part of the surface) is reached at 100 mM Arg, but at 200 mMGly, suggesting that Arg may be the better stabilizer for adalimumab atpH 5.2.

Discussion of PLS Model A—FIG. 16

The final response surface shown for PLS Model A is for the effect ofNaCl and PS 80 (FIG. 16). It shows that the stability of adalimumabdecreases upon addition of NaCl, especially above 100 mM. Meanwhile, PS20 provides significant stability when used above 0.04%.

The Second PLS Model (PLS Model B)

The second PLS model (PLS Model B) used the monomer content at t1 and att2 as the endpoints. The model employed four PCs and had a correlationcoefficient for the calibration set of 0.82 and a r-value of 0.67 forthe validation set. It was a quadratic model including pH-buffer andbuffer-buffer interaction terms. In terms of model quality, this iscomparable to the first PLS Model A described above.

TABLE K (L) PLS “MODEL B” CORRELATION COEFFICIENTS Factor r-value pH−0.086 protein +0.030 citrate −0.079 phos −0.157 succinate +0.060histidine +0.185 acetate +0.063 glycine +0.126 arginine +0.150 sorbitol+0.025 trehalose +0.006 mannitol +0.014 NaCl −0.215 F68 −0.044 PS 20−0.028 PS 80 +0.227 EDTA −0.097 Met +0.096

The endpoints for PLS Model B are the total monomer contents at both t1and t2. Therefore, one will wish to maximize these values. This meansthat stabilizers with have positive correlation coefficients anddestabilizers will display negative r-values (Table K). As with theprevious model, citrate, phosphate, and NaCl are significantdestabilizers. On the other hand, His, Gly Arg, and PS 20 are potentstabilizers. In this model, trehalose, sorbitol and mannitol have verylittle effect. The primary differences are that pH is now a significantfactor and that EDTA is a significant destabilizer, while Met appears tobe a stabilizer as well.

Discussion of PLS Model B—FIG. 17

This model suggests that addition of citrate has little effect onstability if phosphate is absent (view the back edge of the responsesurface of FIG. 5). On the other hand, added phosphate does decreasemonomer content (view the right hand edge) and the combination is evenmore destabilizing (FIG. 5). Thus, the citrate-phosphate buffercombination is not effective at stabilizing adalimumab, contrary to whatis taught by the '583 patent. The destabilizing effect of phosphate isabout three-fold greater than for citrate according to this model.

Discussion of PLS Model B—FIG. 18

The use of His at low pH has little or detrimental effects (FIG. 18[6\).However, when employed at pH 5.2 or above, the His provides asignificant increase in stability (as measured by monomer content bySEC).

Discussion of PLS Model 6—FIG. 19

The Response Surface for Gly and Arg is Shown in FIG. 19. Including Bothstabilizers at high concentrations would be beneficial for stability,but impractical for tonicity reasons. It does appear that Arg is themore potent stabilizer in this model compared to Gly, where a 75 mMconcentration of Arg has the same effect as ˜120 mM Gly. The modelindicates either one alone would work well, or that a combination wouldbe effective as well.

Discussion of PLS Model B—FIG. 20

The PLS model B shows a modest effect of mannitol on stability, whereasPS 80 is an effective stabilizer above concentrations near 0.05% (FIG.20). Thus, one could conclude from this data that a stable formulationcould be comprised of 240 mM mannitol and 0.1% PS 80 at pH 5.2.

Discussion of PLS Model 6—FIG. 21

Throughout the project, it appears that NaCl is a destabilizer ofadalimumab, especially when the concentration reach 100 mM or above, asshown in this response surface (FIG. 21). While only a few formulationswere tested that included EDTA, it appears that this excipient isdestabilizing, unless the concentration were ˜0.1%. We also note thatthe effect of Met was favorable with respect to stability, but it didnot prove to be a significant effect, probably because relatively fewexamples were evaluated.

Discussion of PLS Model B—FIG. 22

The final response surface from the PLS Model B to be considered is theeffect of succinate and His (FIG. 22). The model did include allrelevant buffer-buffer interactions. This surface shows that succinatehas little or even deleterious effects on its own (see the front edge ofthe plot). However, in conjunction with His it proves to increase theoverall stability (e.g., note that back edge of the surface). Therefore,a His-succinate buffer system appear to be the most favorable of all ofthe buffer combinations tested to date.

The Third PLS Model (PLS Model C)

The third PLS model C used the difference in percent purity by RP HPLCat t1 as the endpoint. The model employed two PCs and had a correlationcoefficient for the calibration set of 0.86 and a r-value of 0.67 forthe validation set. It was a quadratic model including pH-buffer andbuffer-buffer interaction terms. In terms of model quality, this is verysimilar to the previous model.

TABLE L PLS “MODEL C” CORRELATION COEFFICIENTS Factor r-value pH −0.115protein −0.139 citrate +0.014 phosphate +0.084 succinate −0.051histidine −0.075 acetate +0.159 glycine −0.096 arginine −0.045 sorbitol+0.029 trehalose +0.020 mannitol −0.060 NaCl +0.068 F68 −0.047 PS 20−0.067 PS 80 −0.028 EDTA +0.099 Met −0.015

PLS Model C demonstrates that RP HPLC is stability-indicating, eventhough the sensitivity may be less than for SEC. The model finds thatboth phosphate and citrate are destabilizing, with the effect ofphosphate being statistically significant (Table LI). Likewise, acetateis a strong destabilizer as is EDTA. Both Gly and Arg are shown to bestabilizers, but the effects are not deemed to be statisticallysignificant. Only His was found to be a significant stabilizer (alongwith protein concentration).

Discussion of PLS Model C—FIG. 23

The response surface for citrate and phosphate at pH 5.2 is shown inFIG. 23[11]. Both buffers are destabilizing (follow the front andleft-hand edges of the plot). Above concentrations of ˜10 mM, thecombination becomes quite destabilizing. Overall, phosphate is predictedto be more destabilizing according to this model (FIG. 11).

Discussion of PLS Model C—FIG. 24

As seen in previous models, the stability of adalimumab decreases as thepH is reduced to less than 5.0 (FIG. 12). In this model the stabilizingeffect of His is seen across all pH values, but is most pronounced whenthe pH is lower.

Discussion of PLS Model C—FIG. 25

The effects of Gly and Arg are seen in FIG. 25. Both excipients decreaseloss of purity as the concentration increases and they are predicted tobe roughly equipotent, as judged by the slopes along the edges of theresponse surface. Otherwise, it appears that it takes less Arg (75 mM)to achieve optimal loss of purity (the blue region of the graph) thanfor Gly (˜200 mM).

Discussion of PLS Model C—FIG. 26

The effect of mannitol and PS 80 is seen in the response surface in FIG.26[14]. It is clear that chemical stability is greatly improved byadding PS 80, especially at concentrations above 0.04%. Meanwhile,mannitol is also stabilizing, but even 240 mM mannitol has less effectthan a small about of the surfactant.

Discussion of PLS Model C—FIG. 27

While mannitol is believed to be a stabilizer in the Humira®formulation, NaCl is clearly a destabilizer, both in this model (SeeFIG. 27[15]), and in previous PLS models. The effect is substantial whenthe NaCl concentration exceeds 75 mM or so.

Discussion of PLS Model C—FIG. 28

The final response surface from PLS Model C is seen in FIG. 28[16]describing the effects of pH and protein concentration. As seen before,the stability is best when the pH is above 4.8 or 5.0. As for theprotein effect, this model predicts that the stability, based on RPHPLC, is better at higher protein concentrations. A similar trend,albeit a fairly weak one, was seen for the SEC data (monomer content att1 and t2). Therefore, it may be possible to achieve similar stabilityprofiles at concentrations at 100 mg/ml as one could obtain at 50 mg/ml.

Summary of Findings for Blocks a Through H

The formulation studies in Blocks A through H evaluated adalimumabformulations stored at elevated temperature and held for either one weekat 40° C. or for two weeks at 25° C. The stability was monitored usingSEC, RP H PLC, cIEF and CE-SDS.

The optimal pH appears to be 5.2±0.2. Of all of the buffer compositionstested, the citrate-phosphate combination is inferior to nearly anyother buffer system evaluated, hence an important aspect of the presentinvention is the avoidance of this combined buffer system altogether.The best single buffer appears to be His, while a His-succinate bufferalso offers very good stability. Even buffer-free systems, which rely onthe ability of the protein to buffer the formulation, appear to haveacceptable stability profiles under accelerated stress conditions.

Of all of the stabilizers/tonicity modifiers evaluated, both Arg and Glyelicit very good stabilization of adalimumab. They both work better thanmannitol. Mannitol does appear to be a stabilizer, however we havediscovered that if used it should be at the highest possibleconcentrations, but in any event exceeding about 150 mM, ad mostpreferably at or exceeding about 200 mM. By comparison, NaCl is clearlya destabilizer, especially when the concentrations exceed 75-100 mM;hence, NaCl, if present should be controlled to levels below about 75mM. Other polyols, such as sorbitol and trehalose, appear to work aboutas well as mannitol and therefore may be substituted for mannitol ifdesired.

Surprisingly, polysorbate 80 (PS 80) provides significant protectionagainst thermal stress. While the mechanism of stabilization is notknown, it appears that other surfactants tested (PS 20 and F-68), do notappear to be nearly as effective as PS 80. Hence the selection of PS80versus PS 20 is a preferred feature of the present invention.Formulations according to the present invention preferably containcontain at least 0.04% (w/v) PS 80.

Based on the findings in the formulation studies of Blocks A through H,the following are particularly preferred adalimumab formulationsaccording to the present invention.

TABLE M SELECTED FORMULATIONS Form His succinate Gly Arg mannitol NaClPS 80 No pH (mM) (mM) (mM) (mM) (mM) (mM) (wt %) A 5.2 30 0 240 0 0 00.1 B 5.2 30 0 240 0 0 0 0.02 C 5.2 30 0 0 0 240 0 0.1 D 5.2 30 15 0 0220 0 0.1 E 5.2 30 0 90 0 150 0 0.1 F 5.2 30 0 240 0 0 0 0 G 5.2 20 0 00 240 0 0 H 5.4 30 0 240 0 0 0 0.02 I 5.2 30 0 120 80 0 0 0.1 J 5.2 3015 90 80 0 0 0.1 K 5.2 30 0 0 0 240 0 0.1 L 5.2 30 0 0 50 160 0 0.1 M5.2 30 0 90 100 0 0 0.1 N 5.2 20 0 120 90 0 0 0.1 O 5.4 30 0 120 80 0 00.1 P 5.2 30 0 120 0 0 50 0.01 Q 5.2 30 0 0 0 240 0 0.02

Additional Components of the Provided Pharmaceutical Compositions

The formulations of the invention may also include other buffers (unlessthey are specifically excluded in the description of the specificembodiments of the invention), tonicity modifiers, excipients,pharmaceutically acceptable carriers and other commonly used inactiveingredients of the pharmaceutical compositions.

A tonicity modifier is a molecule that contributes to the osmolality ofa solution. The osmolality of a pharmaceutical composition is preferablyadjusted to maximize the active ingredient's stability and/or tominimize discomfort to the patient upon administration. It is generallypreferred that a pharmaceutical composition be isotonic with serum,i.e., having the same or similar osmolality, which is achieved byaddition of a tonicity modifier.

In a preferred embodiment, the osmolality of the provided formulationsis from about 180 to about 420 mOsM. However, it is to be understoodthat the osmolality can be either higher or lower as specific conditionsrequire.

Examples of tonicity modifiers suitable for modifying osmolalityinclude, but are not limited to amino acids (not including arginine)(e.g., cysteine, histidine and glycine), salts (e.g., sodium chloride orpotassium chloride) and/or sugars/polyols (e.g., sucrose, sorbitol,maltose, and lactose).

In a preferred embodiment, the concentration of the tonicity modifier inthe formulation is preferably between about 1 mM to about 1 M, morepreferably about 10 mM to about 200 mM. Tonicity modifiers are wellknown in the art and are manufactured by known methods and availablefrom commercial suppliers.

Suitable tonicity modifiers may be present in the compositions of theinvention unless they are specifically excluded in the description ofthe specific embodiments of the invention.

Excipients, also referred to as chemical additives, co-solutes, orco-solvents, that stabilize the polypeptide while in solution (also indried or frozen forms) can also be added to a pharmaceuticalcomposition. Excipients are well known in the art and are manufacturedby known methods and available from commercial suppliers.

Examples of suitable excipients include but are not limited tosugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol,mannitol, maltose, inositol, trehalose, glucose; polymers such as: serumalbumin (bovine serum albumin (BSA), human SA or recombinant HA),dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine,gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC);non-aqueous solvents such as: polyhydric alcohols, (e.g., PEG, ethyleneglycol and glycerol) dimethysulfoxide (DMSO) and dimethylformamide(DMF); amino acids such as: proline, L-serine, sodium glutamic acid,alanine, glycine, lysine hydrochloride, sarcosine and gamma-aminobutyricacid; surfactants such as: Tween®-80 (polysorbate 80), Tween®-20(polysorbate 20), SDS, polysorbates, poloxamers; and miscellaneousexcipients such as: potassium phosphate, sodium acetate, ammoniumsulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide,betaine, CHAPS, monolaurate, 2-O-beta-mannoglycerate or any combinationof the above.

Suitable excipients may be present in the compositions of the inventionunless they are specifically excluded in the description of the specificembodiments of the invention.

The concentration of one or more excipients in a formulation of theinvention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent.

Methods of Treatment

In another embodiment, the invention provides a method of treating amammal comprising administering a therapeutically effective amount ofthe pharmaceutical compositions of the invention to a mammal, whereinthe mammal has a disease or disorder that can be beneficially treatedwith adalimumab.

In a preferred embodiment, the mammal is a human.

Diseases or disorders that can be treated with the provided compositionsinclude but are not limited to rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegener's disease (granulomatosis),Crohn's disease (or inflammatory bowel disease), chronic obstructivepulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia,psoriasis, and atopic dermatitis. Additional diseases or disorders thatcan be treated with the compositions of the present invention includethose described in U.S. Pat. Nos. 6,090,382 and 8,216,583 the relevantportions of which are incorporated herein by reference.

The provided pharmaceutical compositions may be administered to asubject in need of treatment by injection systemically, such as byintravenous injection; or by injection or application to the relevantsite, such as by direct injection, or direct application to the sitewhen the site is exposed in surgery; or by topical application.

In one embodiment, the invention provides a method of treatment and/orprevention of rheumatoid arthritis comprises administering to a mammalin need thereof a therapeutically effective amount of one of theprovided adalimumab compositions.

The therapeutically effective amount of the adalimumab in the providedcompositions will depend on the condition to be treated, the severity ofthe condition, prior therapy, and the patient's clinical history andresponse to the therapeutic agent.

The proper dose can be adjusted according to the judgment of theattending physician such that it can be administered to the patient onetime or over a series of administrations.

In one embodiment, the effective adalimumab amount per adult dose isfrom about 1-500 mg/m², or from about 1-200 mg/m², or from about 1-40mg/m² or about 5-25 mg/m².

Alternatively, a flat dose may be administered, whose amount may rangefrom 2-500 mg/dose, 2-100 mg/dose or from about 10-80 mg/dose.

If the dose is to be administered more than one time per week, anexemplary dose range is the same as the foregoing described dose rangesor lower and preferably administered two or more times per week at a perdose range of 25-100 mg/dose.

In another embodiment, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, containing 80 mgper dose.

The dose can be administered weekly, biweekly, or separated by severalweeks (for example 2 to 8).

In one embodiment, adalimumab is administered at 40 mg by a singlesubcutaneous (SC) injection.

In some instances, an improvement in a patient's condition will beobtained by administering a dose of up to about 100 mg of thepharmaceutical composition one to three times per week over a period ofat least three weeks. Treatment for longer periods may be necessary toinduce the desired degree of improvement. For incurable chronicconditions the regimen may be continued indefinitely. For pediatricpatients (ages 4-17), a suitable regimen may involve administering adose of 0.4 mg/kg to 5 mg/kg of adalimumab, one or more times per week.

In another embodiment, the pharmaceutical formulations of the inventionmay be prepared in a bulk formulation, and as such, the components ofthe pharmaceutical composition are adjusted to be higher than would berequired for administration and diluted appropriately prior toadministration.

The pharmaceutical compositions can be administered as a soletherapeutic or in combination with additional therapies as needed. Thus,in one embodiment, the provided methods of treatment and/or preventionare used in combination with administering a therapeutically effectiveamount of another active agent. The other active agent may beadministered before, during, or after administering the pharmaceuticalcompositions of the present invention. Another active agent may beadministered either as a part of the provided compositions, oralternatively, as a separate formulation.

Administration of the provided pharmaceutical compositions can beachieved in various ways, including parenteral, oral, buccal, nasal,rectal, intraperitoneal, intradermal, transdermal, subcutaneous,intravenous, intra-arterial, intracardiac, intraventricular,intracranial, intratracheal, intrathecal administration, intramuscularinjection, intravitreous injection, and topical application.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, and/or intrathecal. Parenteraladministration can be by bolus injection or continuous infusion.Pharmaceutical compositions for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease®, Genject®, injector pens such as GenPen®,and needleless devices such as MediJector® and BioJector®. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

The provided pharmaceutical compositions can also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions may, if desired, be presented in a vial,pack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. In one embodiment the dispenser devicecan comprise a syringe having a single dose of the liquid formulationready for injection. The syringe can be accompanied by instructions foradministration.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

In addition to the formulations referenced in the formulation studies ofBlocks A through H, the following additional examples are provided asfurther embodiments of the invention, as are the representativeembodiments which are included in Appendices A through C which are to beunderstood as part of this specification.

Example 1 Stabilized Adalimumab Formulation (Simile Buffer) ContainingPolyol; Without Surfactant

A stable aqueous pharmaceutical composition containing adalimumab, usinga single buffer, and substantially free of a surfactant may be preparedas follows:

Each solid formulation component may be weighed to the amount requiredfor a given volume of formulation buffer. These components may then becombined into a beaker or vessel capable of carrying and measuring thegiven volume of formulation buffer. A volume of deionized water equal toapproximately ¾ of the target given formulation buffer may be added tothe beaker, and the components may be solublized through use of amagnetic stir bar. The pH of the buffer may be adjusted to the targetformulation pH using 1 molar sodium hydroxide and/or 1 molar hydrogenchloride. The final formulation buffer volume may then be raised to thetarget volume through the addition of deionized water. The solution maythen be mixed with a magnetic stir bar after final water addition.Adalimumab solution may then be placed in dialysis material housing(such as Thermo Scientific Slide-A-Lyzer MINI Dialysis Unit 10,000MWCO), which may then be placed in contact with the desired formulationbuffer for 12 hours at 4° C. Formulation buffer volume to proteinsolution volume ratio should be no less than 1000:1. The dialysishousing and protein solution it contains may then be placed in a second,equal volume of formulation buffer for an additional 12 hours at 4° C.

Resulting adalimumab solution may then be removed from the dialysismaterial housing, and the concentration of adalimumab may then bedetermined using ultraviolet spectroscopy. Adalimumab concentration maythen be adjusted to the desired level using centrifugation (such asAmicon Ultra 10,000 MWCO Centrifugal Concentrators) and/or dilution withformulation buffer.

A sample composition of the invention is represented in Table 1 below:

TABLE 1 Ingredient concentration Adalimumab (active ingredient) 50 mg/mlMannitol (inactive ingredient) 4% Citrate (pH 5.2) (single buffer) 15 mM

The composition disclosed in Table 1 does not contain a combination ofcitrate and phosphate buffer. It also does not require the presence of asurfactant.

Example 2 Stabilized Adalimumab Formulation (Single Buffer) withoutPolyol or Surfactant

Ingredient concentration Adalimumab (active ingredient) 50 mg/ml Citrate(pH 5.2) 15 mM Glycine (inactive ingredient) 100 mM

Example 3 Stabilized Adalimumab Formulation (Single Buffer) ContainingPolyol without Surfactant

Ingredient concentration Adalimumab (active ingredient) 50 mg/mlMannitol (inactive ingredient) 4% Citrate (pH 5.2) 15 mM

The compositions of examples 2 and 3 have long term stability and do notcontain a combination of citrate and phosphate buffer, and do notrequire the presence of a surfactant.

Example 4 Stabilized Adalimumab Formulation (Simile Buffer) ContainingSurfactant; without Polyol

Ingredient concentration 4A Adalimumab (active ingredient) 50 mg/ml (Nopolyol ingredient) — Histidine Buffer (pH 5.2) (sole buffer) 20 mMGlycine (stabilizer) 50 mM Arginine (stabilizer) 130 mM Polysorbate 800.1 (wt %) (w/v) 4B Adalimumab (active ingredient) 50 mg/ml (No polyolingredient) — Histidine Buffer (pH 5.2) (sole buffer) 20 mM Glycine(stabilizer) 120 mM Arginine (stabilizer) 100 mM Polysorbate 80 0.1 (wt%) (w/v) 4C Adalimumab (active ingredient) 50 mg/ml (No polyolingredient) — Histidine Buffer (pH 5.2) (sole buffer) 10 mM Glycine(stabilizer) 50 mM Arginine (stabilizer) 130 mM Polysorbate 80 0.1 (wt%) (w/v) 4D Adalimumab (active ingredient) 50 mg/ml (No polyolingredient) — Succinate Buffer (pH 5.2) (sole buffer) 20 mM Glycine(stabilizer) 50 mM Arginine (stabilizer) 130 mM Polysorbate 80 0.1 (wt%) (w/v) 4E Adalimumab (active ingredient) 50 mg/ml (No polyolingredient) — Succinate Buffer (pH 5.2) (sole buffer) 20 mM Glycine(stabilizer) 120 mM Arginine (stabilizer) 100 mM Polysorbate 80 0.1 (wt%) (w/v) 4F Adalimumab (active ingredient) 50 mg/ml (No polyolingredient) — Succinate Buffer (pH 5.2) (sole buffer) 10 mM Glycine(stabilizer) 50 mM Arginine (stabilizer) 130 mM Polysorbate 80 0.1 (wt%) (w/v)

The compositions disclosed in Examples 4(a) through 4(f) above willafford stability without need for polyol and without need for a combinedbuffer system. Insofar as the present invention has discovered that thecitrate/phosphate buffer combination required in U.S. Pat. No. 8,216,583is not required for stabilization of adalimumab formulations accordingto the present invention, persons skilled in the art may appreciate, inpracticing examples 4(a) through 4(f), that additional buffers may beemployed in combination with the histidine and succinate buffersdisclosed herein (e.g, acetate, citrate, maleate, tartrate, andphosphate buffers); provided the formulation does not use a buffercombination of citrate and phosphate.

Example 5 Stabilized Adalimumab Formulation (Simile Buffer) ContainingSurfactant; and Polyol

Ingredient Concentration 5A Adalimumab (active ingredient) 50 mg/mlSorbitol 65 mM Histidine Buffer (pH 5.2) (sole buffer) 20 mM Arginine(stabilizer) 130 mM Polysorbate 80 0.1 (wt %) (w/v) 5B Adalimumab(active ingredient) 50 mg/ml Sorbitol 65 mM Succinate Buffer (pH 5.2)(sole buffer) 20 mM Arginine (stabilizer) 130 mM Polysorbate 80 0.1 (wt%) (w/v)

The foregoing description of the exemplary embodiments of the inventionin the block studies A through H, in the examples above, and in theAppendices A through C, are presented only for the purposes ofillustration and description and is not intended to be exhaustive or tolimit the invention to the precise forms disclosed. Many modificationsand variations are possible in light of the above teaching.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

Appendix A Further Representative Embodiments Disclosed in PriorityApplication U.S. Ser. No. 61/698,138

-   -   A. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, a surfactant, and a buffer selected from        the group consisting of citrate, phosphate, succinate,        histidine, tartrate and maleate, wherein said composition has a        pH of about 5 to about 6, and wherein said buffer does not        comprise both of citrate and phosphate.    -   B. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, and a surfactant, wherein said composition        has a pH of about 5 to about 6, and wherein said composition is        substantially free of a buffer.    -   C. The composition of any of embodiments A-B, wherein said        adalimumab is at a concentration from about 20 to about 150        mg/ml.    -   D. The composition of any of embodiments A-C, wherein said        adalimumab is at a concentration from about 20 to about 100        mg/ml.    -   E. The composition of any of embodiments A-D, wherein said        adalimumab is at a concentration from about 30 to about 50        mg/ml.    -   F. The composition of any of embodiments A-E, wherein said        buffer is at a concentration from about 5 mM to about 50 mM.    -   G. The composition of any of embodiments A-F, wherein said        buffer is at a concentration from about 5 mM to about 20 mM.    -   H. The composition of any of embodiments A-G, wherein said        buffer is at a concentration from about 10 mM to about 20 mM.    -   I. The composition of any of embodiments A-G, wherein said        surfactant is a polysorbate.    -   J. The composition of embodiment I, wherein said polysorbate is        polysorbate 80.    -   K. The composition of any of embodiments A-J, wherein said        polyol is a sugar alcohol.    -   L. The composition of embodiment K, wherein said sugar alcohol        is selected from the group consisting of mannitol, sorbitol and        trehalose.    -   M. The composition of embodiment L, wherein said mannitol is at        a concentration from about 1 to 10% weight by volume of the        total composition.    -   N. The composition of any of embodiments L-M, wherein said        mannitol is at a concentration from about 2 to 6% weight by        volume of the total composition.    -   O. The composition of any of embodiments L-N, wherein said        mannitol is at a concentration from about 3 to 5% weight by        volume of the total composition.    -   P. The composition of any of embodiments A-O further comprising        a stabilizer selected from the group consisting of an amino        acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a metal        ion.    -   Q. The composition of embodiment P, wherein said amino acid is        selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   R. The composition of embodiment P, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   S. The composition of embodiment P, wherein said metal ion is        selected from zinc, magnesium and calcium.    -   T. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        mannitol at a concentration from about 1 to 10% weight by        volume, polysorbate 80 at a concentration from about 1 to 50 μM,        and citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH of about 5 to about 5.5, and        wherein said composition is substantially free of phosphate.    -   U. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, and a buffer selected from the group        consisting of citrate, phosphate, succinate, histidine, tartrate        and maleate, wherein said composition has a pH of about 5 to        about 6, and wherein said composition is substantially free of a        surfactant.    -   V. The composition of embodiment U, wherein said adalimumab is        at a concentration from about 20 and about 150 mg/ml.    -   W. The composition of any of embodiments U-V, wherein said        adalimumab is at a concentration from about 20 and about 100        mg/ml.    -   X. The composition of any of embodiments U-V, wherein said        adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   Y. The composition of any of embodiments U-Y, wherein said        buffer is at a concentration from about 5 mM and about 50 mM.    -   Z. The composition of any of embodiments U-Y, wherein said        buffer is at a concentration from about 5 mM and about 20 mM.    -   AA. The composition of any of embodiments U-Z, wherein said        buffer is at a concentration from about 10 mM and about 20 mM.    -   BB. The composition of any of embodiments U-AA, wherein said        polyol is a sugar alcohol.    -   CC. The composition of embodiment BB, wherein said sugar alcohol        is selected from the group consisting of mannitol, sorbitol and        trehalose.    -   DD. The composition of embodiment CC, wherein said mannitol is        at a concentration from about 1 to 10% weight by volume of the        total composition.    -   EE. The composition of any of embodiments CC-DD, wherein said        mannitol is at a concentration from about 2 to 6% weight by        volume of the total composition.    -   FF. The composition of any of embodiments CC-EE, wherein said        mannitol is at a concentration from about 3 to 5% weight by        volume of the total composition.    -   GG. The composition of any of embodiments CC-FF further        comprising a stabilizer selected from the group consisting of an        amino acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a        metal ion.    -   HH. The composition of embodiment GG, wherein said amino acid is        selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   II. The composition of embodiment GG, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   JJ. The composition of embodiment GG, wherein said metal ion is        selected from zinc, magnesium and calcium.    -   KK. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        mannitol at a concentration from about 1 to 10% weight by        volume, and citrate at a concentration from about 5 mM and about        50 mM, wherein said composition has a pH of about 5 to about        5.5, and wherein said composition is substantially free of a        surfactant.    -   LL. A stable aqueous pharmaceutical composition comprising        adalimumab, a buffer, a stabilizer selected from the group        consisting of an amino acid, a salt, ethylenediaminetetraacetic        acid (EDTA) and a metal ion, and wherein said composition has a        pH of about 5 to about 6, and wherein said composition is        substantially free of a polyol.    -   MM. The composition of embodiment LL, wherein said adalimumab is        at a concentration from about 20 and about 150 mg/ml.    -   NN. The composition of any of embodiments LL-MM, wherein said        adalimumab is at a concentration from about 20 and about 100        mg/ml.    -   OO. The composition of any of embodiments LL-NN, wherein said        adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   PP. The composition of any of embodiments LL-OO, wherein said        buffer is at a concentration from about 5 mM and about 50 mM.    -   QQ. The composition of any of embodiments LL-PP, wherein said        buffer is at a concentration from about 5 mM and about 20 mM.    -   RR. The composition of any of embodiments LL-QQ, wherein said        buffer is at a concentration from about 10 mM and about 20 mM.    -   SS. The composition of embodiment LL, wherein said stabilizer is        selected from the group consisting of an amino acid, a salt,        EDTA and a metal ion.    -   TT. The composition of embodiment SS, wherein said amino acid is        selected from the group consisting of glycine, alanine and        arginine.    -   UU. The composition of embodiment SS wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   VV. The composition of embodiment TT, wherein said glycine is at        a concentration from about 20 to about 200 mM.    -   WW. The composition of embodiment W, wherein said glycine is at        a concentration from about 50 to about 200 mM.    -   XX. The composition of embodiment SS, wherein said arginine is        at a concentration from about 1 to about 250 mM.    -   YY. The composition of embodiment XX, wherein said arginine is        at a concentration from about 20 to about 200 mM.    -   ZZ. The composition of embodiment YY, wherein said arginine is        at a concentration from about 20 to about 100 mM.    -   AAA. The composition of embodiment UU, wherein said sodium        chloride is at a concentration from about 5 to about 150 mM.    -   BBB. The composition of embodiment AAA, wherein said sodium        chloride is at a concentration from about 20 to about 140 mM.    -   CCC. The composition of embodiment BBB, wherein said sodium        chloride is at a concentration from about 75 to about 125 mM.    -   DDD. The composition of embodiment UU, wherein said sodium        sulfate is at a concentration from about 5 to about 150 mM.    -   EEE. The composition of embodiment UU, wherein said sodium        chloride is at a concentration from about 20 to about 120 mM.    -   FFF. The composition of embodiment EEE, wherein said sodium        chloride is at a concentration from about 60 to about 100 mM.    -   GGG. The composition of any of embodiments LL-FF further        comprising a surfactant.    -   HHH. The composition of embodiment GGG, wherein said surfactant        is a polysorbate.    -   III. The composition of embodiment HHH, wherein said polysorbate        is polysorbate 80.    -   JJJ. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        glycine at a concentration from about 20 to about 200 mM,        citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH of about 5 to about 5.5, and        wherein said composition is substantially free of a polyol.    -   KKK. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        arginine at a concentration from about 1 to about 250 mM,        citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH from about 5 to about 5.5, and        wherein said composition is substantially free of a polyol.    -   LLL. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        sodium chloride at a concentration from about 5 to about 150 mM,        citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH of about 5 to about 5.5, and        wherein said composition is substantially free of a polyol.    -   MMM. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        sodium chloride at a concentration from about 5 to about 150 mM,        polysorbate 80 at a concentration from about 1 to 50 μM, citrate        at a concentration from about 5 mM and about 50 mM, wherein said        composition has a pH of about 5 to about 5.5, and wherein said        composition is substantially free of a polyol.    -   NNN. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, a surfactant, a stabilizer selected from        the group consisting of an amino acid, a salt,        ethylenediaminetetraacetic acid (EDTA) and a metal ion, and a        buffer selected from the group consisting of citrate, phosphate,        succinate, tartrate and maleate, wherein said composition has a        pH from about 5 to about 6.    -   OOO. The composition of embodiment NNN, wherein the buffer does        not comprise a combination of citrate and phosphate.    -   PPP. The composition of embodiment NNN, wherein said adalimumab        is at a concentration from about 20 and about 150 mg/ml.    -   QQQ. The composition of any of embodiments NNN-PPP, wherein said        adalimumab is at a concentration from about 20 and about 100        mg/ml.    -   RRR. The composition of any of embodiments NNN-QQQ, wherein said        adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   SSS. The composition of any of embodiments NNN-RRR, wherein said        buffer is at a concentration from about 5 mM and about 50 mM.    -   TTT. The composition of any of embodiments NNN-SSS, wherein said        buffer is at a concentration from about 5 mM and about 20 mM.    -   UUU. The composition of any of embodiments NNN-TTT, wherein said        buffer is at a concentration from about 10 mM and about 20 mM.    -   VVV. The composition of any of embodiments NNN-UUU, wherein said        surfactant is a polysorbate.    -   WWW. The composition of embodiment VVV, wherein said polysorbate        is polysorbate 80.    -   XXX. The composition of any of embodiments NNN-WWW, wherein said        polyol is a sugar alcohol.    -   YYY. The composition of embodiment XXX, wherein said sugar        alcohol is selected from the group consisting of mannitol,        sorbitol and trehalose.    -   ZZZ. The composition of embodiment XXX, wherein said mannitol is        at a concentration from about 1 to 10% weight by volume of the        total composition.    -   AAAA. The composition of any of embodiments XXX-ZZZ, wherein        said mannitol is at a concentration from about 2 to 6% weight by        volume of the total composition.    -   BBBB. The composition of any of embodiments YYY-AAAA, wherein        said mannitol is at a concentration from about 3 to 5% weight by        volume of the total composition.    -   CCCC. The composition of any of embodiments NNN-BBBB, wherein        said stabilizer is EDTA.    -   DDDD. The composition of embodiment CCCC, wherein said EDTA is        at a concentration from about 0.01% to about 0.5%.    -   EEEE. The composition of embodiment DDDD, wherein said EDTA is        at a concentration from about 0.05% to about 0.25%.    -   FFFF. The composition of embodiment EEEE, wherein said EDTA is        at a concentration from about 0.08% to about 0.2%.    -   GGGG. The composition of any of embodiments NNN-BBBB, wherein        said stabilizer is methionine.    -   HHHH. The composition of embodiment GGGG, wherein said        methionine is at a concentration from about 1 to about 10 mg/ml.    -   IIII. The composition of embodiment GGGG, wherein said        methionine is at a concentration from about 1 to about 5 mg/ml.    -   JJJJ. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 1 to about 50 μM,        mannitol at a concentration from about 1 to 10% weight by        volume, EDTA at a concentration from about 0.01% to about 0.5%,        citrate at a concentration from about 5 mM and about 50 mM, and        wherein said composition has a pH of about 5 to about 5.5.    -   KKKK. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 1 to about 50 μM,        mannitol at a concentration from about 1 to 10% weight by        volume, methionine at a concentration from about 1 to about 10        mg/ml, citrate at a concentration from about 5 mM and about 50        mM, and wherein said composition has a pH of about 5 to about        5.5.    -   LLLL. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, and a buffer selected from the group        consisting of citrate, phosphate, succinate, tartrate and        maleate, wherein said composition has a pH of about 3.5.    -   MMMM. The composition of embodiment LLLL, wherein said        adalimumab is at a concentration from about 20 and about 150        mg/ml.    -   NNNN. The composition of any of embodiments LLLL-MMMM, wherein        said adalimumab is at a concentration from about 20 and about        100 mg/ml.    -   OOOO. The composition of any of embodiments LLLL-NNNN, wherein        said adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   PPPP. The composition of any of embodiments LLLL-OOOO, wherein        said buffer is at a concentration from about 5 mM and about 50        mM.    -   QQQQ. The composition of any of embodiments LLLL-PPPP, wherein        said buffer is at a concentration from about 5 mM and about 20        mM.    -   RRRR. The composition of any of embodiments LLLL-QQQQ, wherein        said buffer is at a concentration from about 10 mM and about 20        mM.    -   SSSS. The composition of any of embodiments LLLL-RRRR, wherein        said polyol is a sugar alcohol.    -   TTTT. The composition of embodiment SSSS, wherein said sugar        alcohol is selected from the group consisting of mannitol,        sorbitol and trehalose.    -   UUUU. The composition of embodiment TTTT, wherein said mannitol        is at a concentration from about 1 to about 10% weight by volume        of the total composition.    -   VVVV. The composition of any of embodiments TTTT-UUUU, wherein        said mannitol is at a concentration from about 2 to about 6%        weight by volume of the total composition.    -   WWWW. The composition of any of embodiments TTTT-VVVV, wherein        said mannitol is at a concentration from about 3 to about 5%        weight by volume of the total composition.    -   XXXX. The composition of any of embodiments LLLL-WWWW further        comprising a stabilizer selected from the group consisting of an        amino acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a        metal ion.    -   YYYY. The composition of embodiment XXXX, wherein said amino        acid is selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   ZZZZ. The composition of embodiment XXXX, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   AAAAA. The composition of embodiment XXXX, wherein said metal        ion is selected from zinc, magnesium and calcium.    -   BBBBB. The composition of any of embodiments TTTT-AAAAA further        comprising a surfactant.    -   CCCCC. The composition of embodiment BBBBB, wherein said        surfactant is a polysorbate.    -   DDDDD. The composition of embodiment CCCCC, wherein said        polysorbate is polysorbate 80.

Appendix B Further Representative Embodiments Disclosed in PriorityApplication U.S. Ser. No. 61/770,421

-   -   A. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, a surfactant, and a buffer selected from        the group consisting of citrate, phosphate, succinate,        histidine, tartrate and maleate, wherein said composition has a        pH of about 5 to about 6, and wherein said buffer does not        comprise both of citrate and phosphate.    -   B. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, and a surfactant, wherein said composition        has a pH of about 5 to about 6, and wherein said composition is        substantially free of a buffer.    -   C. The composition of any of embodiments A-B, wherein said        adalimumab is at a concentration from about 20 to about 150        mg/ml.    -   D. The composition of any of embodiments A-C, wherein said        adalimumab is at a concentration from about 20 to about 100        mg/ml.    -   E. The composition of any of embodiments A-D, wherein said        adalimumab is at a concentration from about 30 to about 50        mg/ml.    -   F. The composition of any of embodiments A-E, wherein said        buffer is at a concentration from about 5 mM to about 50 mM.    -   G. The composition of any of embodiments A-F, wherein said        buffer is at a concentration from about 5 mM to about 20 mM.    -   H. The composition of any of embodiments A-G, wherein said        buffer is at a concentration from about 10 mM to about 20 mM.    -   I. The composition of any of embodiments A-H, wherein said        surfactant is a polysorbate.    -   J. The composition of embodiment I, wherein said polysorbate is        polysorbate 80.    -   K. The composition of any of embodiments A-J, wherein said        polyol is a sugar alcohol.    -   L. The composition of embodiment K, wherein said sugar alcohol        is selected from the group consisting of mannitol, sorbitol and        trehalose.    -   M. The composition of embodiment L, wherein said mannitol is at        a concentration from about 1 to 10% weight by volume of the        total composition.    -   N. The composition of any of embodiments L-M, wherein said        mannitol is at a concentration from about 2 to 6% weight by        volume of the total composition.    -   O. The composition of any of embodiments L-N, wherein said        mannitol is at a concentration from about 3 to 5% weight by        volume of the total composition.    -   P. The composition of any of embodiments A-O further comprising        a stabilizer selected from the group consisting of an amino        acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a metal        ion.    -   Q. The composition of embodiment P, wherein said amino acid is        selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   R. The composition of embodiment P, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   S. The composition of embodiment P, wherein said metal ion is        selected from zinc, magnesium and calcium.    -   T. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        mannitol at a concentration from about 1 to 10% weight by        volume, polysorbate 80 at a concentration from about 1 to 50 μM,        and citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH of about 5 to about 5.5, and        wherein said composition is substantially free of phosphate.    -   UA stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, and a buffer selected from the group        consisting of citrate, phosphate, succinate, histidine, tartrate        and maleate, wherein said composition has a pH of about 5 to        about 6, and wherein said composition is substantially free of a        surfactant.    -   V. The composition of embodiment U, wherein said adalimumab is        at a concentration from about 20 and about 150 mg/ml.    -   W. The composition of any of embodiments U-V, wherein said        adalimumab is at a concentration from about 20 and about 100        mg/ml.    -   X. The composition of any of embodiments U-W, wherein said        adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   Y. The composition of any of embodiments U-X, wherein said        buffer is at a concentration from about 5 mM and about 50 mM.    -   Z. The composition of any of embodiments U-Y, wherein said        buffer is at a concentration from about 5 mM and about 20 mM.    -   AA. The composition of any of embodiments U-Z, wherein said        buffer is at a concentration from about 10 mM and about 20 mM.    -   BB. The composition of any of embodiments U-AA, wherein said        polyol is a sugar alcohol.    -   CC. The composition of embodiment BB, wherein said sugar alcohol        is selected from the group consisting of mannitol, sorbitol and        trehalose.    -   DD. The composition of embodiment CC, wherein said mannitol is        at a concentration from about 1 to 10% weight by volume of the        total composition.    -   EE. The composition of any of embodiments CC-DD, wherein said        mannitol is at a concentration from about 2 to 6% weight by        volume of the total composition.    -   FF. The composition of any of embodiments CC-EE, wherein said        mannitol is at a concentration from about 3 to 5% weight by        volume of the total composition.    -   GG. The composition of any of embodiments U-FF further        comprising a stabilizer selected from the group consisting of an        amino acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a        metal ion.    -   HH. The composition of embodiment GG, wherein said amino acid is        selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   II. The composition of embodiment GG, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   JJ. The composition of embodiment GG, wherein said metal ion is        selected from zinc, magnesium and calcium.    -   KK. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        mannitol at a concentration from about 1 to 10% weight by        volume, and citrate at a concentration from about 5 mM and about        50 mM, wherein said composition has a pH of about 5 to about        5.5, and wherein said composition is substantially free of a        surfactant.    -   LL. A stable aqueous pharmaceutical composition comprising        adalimumab, a buffer, a stabilizer selected from the group        consisting of an amino acid, a salt, ethylenediaminetetraacetic        acid (EDTA) and a metal ion, and wherein said composition has a        pH of about 5 to about 6, and wherein said composition is        substantially free of a polyol.    -   MM. The composition of embodiment LL, wherein said adalimumab is        at a concentration from about 20 and about 150 mg/ml.    -   NN. The composition of any of embodiments LL-MM, wherein said        adalimumab is at a concentration from about 20 and about 100        mg/ml.    -   OO. The composition of any of embodiments LL-NN, wherein said        adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   PP. The composition of any of embodiments LL-OO, wherein said        buffer is at a concentration from about 5 mM and about 50 mM.    -   QQ. The composition of any of embodiments LL-PP, wherein said        buffer is at a concentration from about 5 mM and about 20 mM.    -   RR. The composition of any of embodiments LL-QQ, wherein said        buffer is at a concentration from about 10 mM and about 20 mM.    -   SS. The composition of embodiment LL, wherein said stabilizer is        selected from the group consisting of an amino acid, a salt,        EDTA and a metal ion.    -   TT. The composition of embodiment TT, wherein said amino acid is        selected from the group consisting of glycine, alanine and        arginine.    -   UU. The composition of embodiment TT, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   VV. The composition of embodiment TT, wherein said glycine is at        a concentration from about 20 to about 200 mM.    -   WW. The composition of embodiment W, wherein said glycine is at        a concentration from about 50 to about 200 mM.    -   XX. The composition of embodiment TT, wherein said arginine is        at a concentration from about 1 to about 250 mM.    -   YY. The composition of embodiment XX, wherein said arginine is        at a concentration from about 20 to about 200 mM.    -   ZZ. The composition of embodiment YY, wherein said arginine is        at a concentration from about 20 to about 100 mM.    -   AAA. The composition of embodiment UU, wherein said sodium        chloride is at a concentration from about 5 to about 150 mM.    -   BBB. The composition of embodiment AAA, wherein said sodium        chloride is at a concentration from about 20 to about 140 mM.    -   CCC. The composition of embodiment AAA, wherein said sodium        chloride is at a concentration from about 75 to about 125 mM.    -   DDD. The composition of embodiment UU, wherein said sodium        sulfate is at a concentration from about 5 to about 150 mM.    -   EEE. The composition of embodiment UU, wherein said sodium        chloride is at a concentration from about 20 to about 120 mM.    -   FFF. The composition of embodiment EEE, wherein said sodium        chloride is at a concentration from about 60 to about 100 mM.    -   GGG. The composition of any of embodiments LL-FF further        comprising a surfactant.    -   HHH. The composition of embodiment GGG, wherein said surfactant        is a polysorbate.    -   III. The composition of embodiment HHH, wherein said polysorbate        is polysorbate 80.    -   JJJ. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        glycine at a concentration from about 20 to about 200 mM,        citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH of about 5 to about 5.5, and        wherein said composition is substantially free of a polyol.    -   KKK. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        arginine at a concentration from about 1 to about 250 mM,        citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH from about 5 to about 5.5, and        wherein said composition is substantially free of a polyol.    -   LLL. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        sodium chloride at a concentration from about 5 to about 150 mM,        citrate at a concentration from about 5 mM and about 50 mM,        wherein said composition has a pH of about 5 to about 5.5, and        wherein said composition is substantially free of a polyol.    -   MMM. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        sodium chloride at a concentration from about 5 to about 150 mM,        polysorbate 80 at a concentration from about 1 to 50 μM, citrate        at a concentration from about 5 mM and about 50 mM, wherein said        composition has a pH of about 5 to about 5.5, and wherein said        composition is substantially free of a polyol.    -   NNN. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, a surfactant, a stabilizer selected from        the group consisting of an amino acid, a salt,        ethylenediaminetetraacetic acid (EDTA) and a metal ion, and a        buffer selected from the group consisting of citrate, phosphate,        succinate, tartrate and maleate, wherein said composition has a        pH from about 5 to about 6.    -   OOO. The composition of embodiment NNN, wherein the buffer does        not comprise a combination of citrate and phosphate.    -   PPP. The composition of embodiment NNN, wherein said adalimumab        is at a concentration from about 20 and about 150 mg/ml.    -   QQQ. The composition of any of embodiments NNN-PPP, wherein said        adalimumab is at a concentration from about 20 and about 100        mg/ml.    -   RRR. The composition of any of embodiments NNN-QQQ, wherein said        adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   SSS. The composition of any of embodiments NNN-RRR, wherein said        buffer is at a concentration from about 5 mM and about 50 mM.    -   TTT. The composition of any of embodiments NNN-SSS, wherein said        buffer is at a concentration from about 5 mM and about 20 mM.    -   UUU. The composition of any of embodiments NNN-TTT, wherein said        buffer is at a concentration from about 10 mM and about 20 mM.    -   VVV. The composition of any of embodiments NNN-UUU, wherein said        surfactant is a polysorbate.    -   WWW. The composition of embodiment VVV, wherein said polysorbate        is polysorbate 80.    -   XXX. The composition of any of embodiments NNN-WWW, wherein said        polyol is a sugar alcohol.    -   YYY. The composition of embodiment XXX, wherein said sugar        alcohol is selected from the group consisting of mannitol,        sorbitol and trehalose.    -   ZZZ. The composition of embodiment YYY, wherein said mannitol is        at a concentration from about 1 to 10% weight by volume of the        total composition.    -   AAAA. The composition of any of embodiments YYY-ZZZ, wherein        said mannitol is at a concentration from about 2 to 6% weight by        volume of the total composition.    -   BBBB. he composition of any of embodiments YYY-AAAA, wherein        said mannitol is at a concentration from about 3 to 5% weight by        volume of the total composition.    -   CCCC. The composition of any of embodiments NNN-BBBB, wherein        said stabilizer is EDTA.    -   DDDD. The composition of embodiment CCCC, wherein said EDTA is        at a concentration from about 0.01% to about 0.5%.    -   EEEE. The composition of embodiment DDDD, wherein said EDTA is        at a concentration from about 0.05% to about 0.25%.    -   FFFF. The composition of embodiment EEEE, wherein said EDTA is        at a concentration from about 0.08% to about 0.2%.    -   GGGG. The composition of any of embodiments NNN-BBBB, wherein        said stabilizer is methionine.    -   HHHH. The composition of embodiment GGGG, wherein said        methionine is at a concentration from about 1 to about 10 mg/ml.    -   IIII. The composition of embodiment GGGG, wherein said        methionine is at a concentration from about 1 to about 5 mg/ml.    -   JJJJ. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 1 to about 50 μM,        mannitol at a concentration from about 1 to 10% weight by        volume, EDTA at a concentration from about 0.01% to about 0.5%,        citrate at a concentration from about 5 mM and about 50 mM, and        wherein said composition has a pH of about 5 to about 5.5.    -   KKKK. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 1 to about 50 μM,        mannitol at a concentration from about 1 to 10% weight by        volume, methionine at a concentration from about 1 to about 10        mg/ml, citrate at a concentration from about 5 mM and about 50        mM, and wherein said composition has a pH of about 5 to about        5.5.    -   LLLL. A stable aqueous pharmaceutical composition comprising        adalimumab, a polyol, and a buffer selected from the group        consisting of citrate, phosphate, succinate, tartrate and        maleate, wherein said composition has a pH of about 3.5.    -   MMMM. The composition of embodiment LLLL, wherein said        adalimumab is at a concentration from about 20 and about 150        mg/ml.    -   NNNN. The composition of any of embodiments LLLL-MMMM, wherein        said adalimumab is at a concentration from about 20 and about        100 mg/ml.    -   OOOO. The composition of any of embodiments LLLL-NNNN, wherein        said adalimumab is at a concentration from about 20 and about 40        mg/ml.    -   PPPP. The composition of any of embodiments LLLL-OOOO, wherein        said buffer is at a concentration from about 5 mM and about 50        mM.    -   QQQQ. The composition of any of embodiments LLLL-PPPP, wherein        said buffer is at a concentration from about 5 mM and about 20        mM.    -   RRRR. The composition of any of embodiments LLLL-QQQQ, wherein        said buffer is at a concentration from about 10 mM and about 20        mM.    -   SSSS. The composition of any of embodiments LLLL-RRRR, wherein        said polyol is a sugar alcohol.    -   TTTT. The composition of embodiment SSSS, wherein said sugar        alcohol is selected from the group consisting of mannitol,        sorbitol and trehalose.    -   UUUU. The composition of embodiment TTTT, wherein said mannitol        is at a concentration from about 1 to about 10% weight by volume        of the total composition.    -   VVVV. The composition of any of embodiments TTTT-UUUU, wherein        said mannitol is at a concentration from about 2 to about 6%        weight by volume of the total composition.    -   WWWW. The composition of any of embodiments TTTT-VVVV, wherein        said mannitol is at a concentration from about 3 to about 5%        weight by volume of the total composition.    -   XXXX. The composition of any of embodiments LLLL-WWWW further        comprising a stabilizer selected from the group consisting of an        amino acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a        metal ion.    -   YYYY. The composition of embodiment XXXX, wherein said amino        acid is selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   ZZZZ. The composition of embodiment XXXX, wherein said salt is        selected from the group consisting of sodium chloride and sodium        sulfate.    -   AAAAA. The composition of embodiment XXXX, wherein said metal        ion is selected from zinc, magnesium and calcium.    -   BBBBB. The composition of any of embodiments LLLL-AAAAA further        comprising a surfactant.    -   CCCCC. The composition of embodiment BBBBB, wherein said        surfactant is a polysorbate.    -   DDDDD. The composition of embodiment CCCCC, wherein said        polysorbate is polysorbate 80.    -   EEEEE. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 1 to about 50 μM;        polyol selected from sorbitol, mannitol or trehalose at a        concentration from about 1 to about 10% weight by volume, and at        least one amino acid stabilizer selected from the group        consisting of (a) arginine at a concentration from about 1 to        about 250 mg/ml and (b) glycine at a concentration of about 20        to 200 mg/ml, and histidine buffer or succinate buffer at a        concentration from about 5 mM and about 50 mM, and wherein said        composition has a pH of about 5 to about 5.5.    -   FFFFF. The composition of embodiment EEEEE wherein the polyol is        sorbitol, and the composition is free or substantially free of        any citrate/phosphate buffer combination, and the formulation        comprises arginine or glycine, but not both.    -   GGGGG. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 1 to about 50 μM,        arginine at a concentration from about 1 to about 250 mg/ml,        glycine at a concentration of about 20 to 200 mg/ml, and        histidine buffer or succinate buffer at a concentration from        about 5 mM and about 50 mM, and wherein said composition has a        pH of about 5 to about 5.5 and said composition is free or        substantially free of polyol.    -   HHHHH. The composition of embodiment GGGGG wherein the the        composition is free or substantially free of any        citrate/phosphate buffer combination.

Appendix C Further Representative Embodiments Disclosed in PriorityApplication U.S. Ser. No. 61/769,581

-   -   A. A stable aqueous pharmaceutical composition comprising        adalimumab and a single buffer.    -   B. The composition of embodiment A, wherein said single buffer        is selected from the group consisting of succinate, histidine,        citrate, phosphate, tartrate and maleate.    -   C. The composition of any of the preceding embodiments, wherein        said composition has a pH of about 5 to about 6.    -   D. The composition of any of the preceding embodiments, wherein        said adalimumab contained in said pharmaceutical compositions        does not lose more than 20% of its activity relative to activity        of the composition at the beginning of storage.    -   E. The composition of any of the preceding embodiments, further        comprising a surfactant.    -   F. The composition of embodiment E, wherein said surfactant is a        polysorbate.    -   G. The composition of embodiment F wherein said polysorbate is        polysorbate 80.    -   H. The composition of any of the preceding embodiments, further        comprising a polyol.    -   I. The composition of embodiment H, wherein said polyol is a        sugar alcohol.    -   J. The composition of embodiment I, wherein said sugar alcohol        is sorbitol.    -   K. The composition of any of the preceding embodiments, further        comprising a sugar.    -   L. The composition of embodiment K, wherein said sugar is        selected from the group consisting of sucrose and trehalose.    -   M. The composition of any of the preceding embodiments, wherein        said adalimumab is at a concentration from about 20 to about 150        mg/ml.    -   N. The composition of any of the preceding embodiments, wherein        said buffer is at a concentration from about 5 mM to about 50        mM.    -   O. The composition of any of embodiments A-N further comprising        a stabilizer selected from the group consisting of an amino        acid, a salt, ethylenediaminetetraacetic acid (EDTA) and a metal        ion.    -   P. The composition of embodiment O, wherein said amino acid is        selected from the group consisting of glycine, alanine,        glutamate, arginine and methionine.    -   Q. The composition of embodiment O, wherein said metal ion is        selected from zinc, magnesium and calcium.    -   R. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 0.01% w/v to 0.5%        w/v by weight of the total formulation, and succinate at a        concentration from about 5 mM and about 50 mM, wherein said        composition has a pH of about 5 to about 5.5, and wherein said        composition is substantially free of any other buffers.    -   S. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 0.01% w/v to 0.5%        w/v by weight of the total formulation, and histidine at a        concentration from about 5 mM and about 50 mM, wherein said        composition has a pH of about 5 to about 5.5, and wherein said        composition is substantially free of any other buffers.    -   T. A stable aqueous pharmaceutical composition comprising        adalimumab at a concentration from about 20 and about 150 mg/ml,        polysorbate 80 at a concentration from about 0.01% w/v to 0.5%        w/v by weight of the total formulation, and tartrate at a        concentration from about 5 mM and about 50 mM, wherein said        composition has a pH of about 5 to about 5.5, and wherein said        composition is substantially free of any other buffers.    -   U. A method of treating a mammal comprising administering to        said mammal a therapeutically effective amount of the        composition of any of preceding embodiments, wherein said mammal        has a disease or disorder that can be beneficially treated with        adalimumab.    -   V. The method of embodiment U, wherein said disease or disorder        is selected from the group consisting of rheumatoid arthritis,        psoriatic arthritis, ankylosing spondylitis, Wegener's disease        (granulomatosis), Crohn's disease (or inflammatory bowel        disease), chronic obstructive pulmonary disease (COPD),        Hepatitis C, endometriosis, asthma, cachexia, psoriasis, and        atopic dermatitis.

What is claimed is:
 1. A stable aqueous pharmaceutical compositioncomprising: a) adalimumab; b) a buffer; c) a tonicity modifier; d)mannitol; and e) a surfactant, wherein said buffer does not comprise acombination of citrate and phosphate, said tonicity modifier is a salt,and the composition has a pH of about 5 to about
 6. 2. The compositionof claim 1, wherein the composition has osmolality of about 180 to 420mOsM; the composition is suitable for administration to a subject as asingle dose; and the dose contains about 40 mg of adalimumab.
 3. Thecomposition of claim 1, wherein the pH is about 5.2.
 4. The compositionof claim 3, wherein the composition has osmolality of about 180 to 420mOsM; the composition is suitable for administration to a subject as asingle dose; and the dose contains about 40 mg of adalimumab.
 5. Astable aqueous pharmaceutical composition comprising: a) adalimumab; b)a buffer; c) a tonicity modifier; d) mannitol; and e) a surfactant,wherein said buffer does not comprise a combination of citrate andphosphate, said tonicity modifier is sodium chloride, and thecomposition has a pH of about 5 to about
 6. 6. The composition of claim5, wherein the composition has osmolality of about 180 to 420 mOsM; thecomposition is suitable for administration to a subject as a singledose; and the dose contains about 40 mg of adalimumab.
 7. Thecomposition of claim 5, wherein the pH is about 5.2.
 8. The compositionof claim 7, wherein the composition has osmolality of about 180 to 420mOsM; the composition is suitable for administration to a subject as asingle dose; and the dose contains about 40 mg of adalimumab.
 9. Astable aqueous pharmaceutical composition comprising: a) adalimumab; b)a buffer; c) a tonicity modifier; d) mannitol; and e) polysorbate 80,wherein said buffer does not comprise a combination of citrate andphosphate, said tonicity modifier is a salt, and the composition has apH of about 5 to about
 6. 10. The composition of claim 9, wherein thecomposition has osmolality of about 180 to 420 mOsM; the composition issuitable for administration to a subject as a single dose; and the dosecontains about 40 mg of adalimumab.
 11. The composition of claim 9,wherein the pH is about 5.2.
 12. The composition of claim 11, whereinthe composition has osmolality of about 180 to 420 mOsM; the compositionis suitable for administration to a subject as a single dose; and thedose contains about 40 mg of adalimumab.
 13. A stable aqueouspharmaceutical composition comprising: a) adalimumab; b) a buffer; c) atonicity modifier; d) mannitol; and e) polysorbate 80, wherein saidbuffer does not comprise a combination of citrate and phosphate, saidtonicity modifier is sodium chloride, and the composition has a pH ofabout 5 to about
 6. 14. The composition of claim 13, wherein thecomposition has osmolality of about 180 to 420 mOsM; the composition issuitable for administration to a subject as a single dose; and the dosecontains about 40 mg of adalimumab.
 15. The composition of claim 13,wherein the pH is about 5.2.
 16. The composition of claim 15, whereinthe composition has osmolality of about 180 to 420 mOsM; the compositionis suitable for administration to a subject as a single dose; and thedose contains about 40 mg of adalimumab.